TOMM34 peptides and vaccines including the same

ABSTRACT

The present invention provides isolated peptides or the fragments derived from SEQ ID NO: 42, which bind to an HLA antigen and induce cytotoxic T lymphocytes (CTL). The peptides may include one of the above mentioned amino acid sequences with substitution, deletion, or addition of one, two, or several amino acids sequences. The present invention also provides pharmaceutical compositions including these peptides. The peptides of this invention can be used for treating cancer.

PRIORITY

The present application claims the benefit of U.S. ProvisionalApplication No. 61/419,181 filed on Dec. 2, 2010, the entire contents ofwhich are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to the field of biological science, morespecifically to the field of cancer therapy. In particular, the presentinvention relates to novel peptides that are extremely effective ascancer vaccines, and drugs for treating and preventing tumors.

BACKGROUND ART

It has been demonstrated that CD8-positive CTLs recognize epitopepeptides derived from tumor-associated antigens (TAAs) on the majorhistocompatibility complex (MHC) class I molecule, and then kill thetumor cells. Since the discovery of melanoma antigen (MAGE) family asthe first example of TAAs, many other TAAs have been discovered throughimmunological approaches (NPLs 1, 2), and some of these TAAs are now inthe process of clinical development as immunotherapeutic targets.

Favorable TAA is indispensable for proliferation and survival of cancercells, as a target for immunotherapy, because the use of such TAAs mayminimize the well-described risk of immune escape of cancer cellsattributable to deletion, mutation, or down-regulation of TAAs as aconsequence of therapeutically driven immune selection. Accordingly, theidentification of new TAAs capable of inducing potent and specificanti-tumor immune responses warrants further development and thusclinical application of peptide vaccination strategies for various typesof cancer is ongoing (NPLs 3 to 10). To date, several clinical trialsusing these TAA derived peptides have been reported. Unfortunately, manyof the current cancer vaccine trials have shown only a low objectiveresponse rate (NPLs 11 to 13). Accordingly, there remains a need for newTAAs as immunotherapeutic targets.

The TOMM34 gene (GenBank Accession No. NM_006809), translocase of outermitochondrial membrane 34, has been identified from human EST and cDNAdatabases and predicted to encode a protein containing degeneratedtetratricopeptide repeat (TPR) motifs (NPL 14). The protein encoded bythis gene is involved in the import of precursor proteins intomitochondria. This protein has a chaperone-like activity, binding themature portion of unfolded proteins and aiding their import intomitochondria (NPL 15).

In a recent study, it was indicated that TOMM34 is frequentlyup-regulated in colorectal cancer by gene-expression profile analysisusing cDNA microarray consisting of 23040 genes. Furthermore, knockdownof TOMM34 by siRNA in colon cancer cell lines attenuated the growth ofcolon cancer cells (NPL 16).

CITATION LIST Patent Literature

-   [PTL 1] PCT/JP2006/314947

Non Patent Literature

-   [NPL 1] Boon T, Int J Cancer 1993, 54(2): 177-80-   [NPL 2] Boon T & van der Bruggen P, J Exp Med 1996, 183(3): 725-9-   [NPL 3] Harris C C, J Natl Cancer Inst 1996, 88(20): 1442-55-   [NPL 4] Butterfield L H et al., Cancer Res 1999, 59(13): 3134-42-   [NPL 5] Vissers J L et al., Cancer Res 1999, 59(21): 5554-9-   [NPL 6] van der Burg S H et al., J Immunol 1996, 156(9): 3308-14-   [NPL 7] Tanaka F et al., Cancer Res 1997, 57(20): 4465-8-   [NPL 8] Fujie T et al., Int J Cancer 1999, 80(2): 169-72-   [NPL 9] Kikuchi M et al., Int J Cancer 1999, 81(3): 459-66-   [NPL 10] Oiso M et al., Int J Cancer 1999, 81(3): 387-94-   [NPL 11] Belli F et al., J Clin Oncol 2002, 20(20): 4169-80-   [NPL 12] Coulie P G et al., Immunol Rev 2002, 188: 33-42-   [NPL 13] Rosenberg S A et al., Nat Med 2004, 10(9): 909-15-   [NPL 14] Nuttal S D et al., DNA Cell Biol. 1997 September;    16(9):1067-74-   [NPL 15] Mukhopadhyay A et al., Arch Biochem Biophys. 2002 Apr. 1;    400(1):97-104-   [NPL 16] Shimokawa et al., Int J Oncol. 2006 August; 29(2):381-6

SUMMARY OF INVENTION

The present invention is based, at least in part, on the discovery ofnovel peptides that may serve as suitable targets of immunotherapy.Because TAAs are generally perceived by the immune system as “self” andtherefore often have no immunogenicity, the discovery of appropriatetargets is of extreme importance. As noted above, TOMM34 (for example,SEQ ID NOs: 41 and 42, also indicated in GenBank Accession No.NM_006809) has been identified as up-regulated in cancers, including,but are not limited to, acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), bladder cancer, breast cancer, cervicalcancer, colorectal cancer, esophagus cancer, liver cancer, osteosarcoma,prostate cancer, renal carcinoma, small cell lung cancer (SCLC),non-small cell lung cancer (NSCLC) and soft tissue tumor. Thus, thepresent invention focuses on TOMM34 as an appropriate cancer marker anda candidate for the target of immunotherapy.

In the course of the present invention, specific epitope peptides of thegene products of TOMM34 that possess the ability to induce CTLs specificto TOMM34 were identified. As discussed in greater detail below,peripheral blood mononuclear cells (PBMCs) obtained from a healthy donorwere stimulated using HLA-A*0201 binding candidate peptides derived fromTOMM34. CTL lines were then established with specific cytotoxicityagainst the HLA-A2 positive target cells pulsed with each of candidatepeptides. The results herein demonstrate that these peptides are HLA-A2restricted epitope peptides that may induce potent and specific immuneresponses against cells expressing TOMM34. These results furtherindicate that TOMM34 is strongly immunogenic and the epitopes thereofare effective targets for tumor immunotherapy.

Accordingly, it is an object of the present invention to provideisolated peptides that bind to HLA antigen and include the amino acidsequence of TOMM34 (SEQ ID NO: 42) or the immunologically activefragments thereof. These peptides are expected to have CTL inducibilityand, thus, can be used to induce CTL in vitro or ex vivo, or to beadministered to a subject for inducing immune responses against cancers,examples of which include, but are not limited to AML, CML, bladdercancer, breast cancer, cervical cancer, colorectal cancer, esophaguscancer, liver cancer, osteosarcoma, prostate cancer, renal carcinoma,SCLC, NSCLC and soft tissue tumor. Preferred those peptides arenonapeptides or decapeptides, and, more preferably, a nonapeptide ordecapeptide consisting of an amino acid sequence selected from among SEQID NOs: 1 to 20 and 22 to 40. Of these, the peptides having an aminoacid sequence selected from among SEQ TD NOs: 1, 5, 31 and 32 showedparticularly strong CTL inducibility and thus are particularlypreferred.

The present invention also contemplates modified peptides having anamino acid sequence of an immunologically active fragment of TOMM34 inwhich one, two or more amino acids are substituted, deleted, inserted oradded, so long as the modified peptides retain the requisite CTLinducibility of the original unmodified peptide. Of these, peptidehaving an amino sequence of SEQ ID NO: 1, 5, 31 or 32 in which one, twoor more amino acids are substituted, deleted, inserted or added areparticularly preferred.

The present invention further encompasses isolated polynucleotidesencoding any peptides of the present invention. These polynucleotidescan be used to induce or prepare APCs having CTL inducibility. Like theabove-described peptides of the present invention, such APCs can beadministered to a subject for inducing immune responses against cancers.

When administered to a subject, the present peptides are presented onthe surface of APCs so as to induce CTLs targeting the respectivepeptides. Therefore, one object of the present invention is to providecompositions or agents including any peptides or polynucleotidesprovided by the present invention for inducing CTL. Such compositions oragents, including any peptides or polynucleotides, can be used for thetreatment and/or prophylaxis of cancers or the prevention of apostoperative recurrence of cancer, examples of which include, but arenot limited to, AML, CML, bladder cancer, breast cancer, cervicalcancer, colorectal cancer, esophagus cancer, liver cancer, osteosarcoma,prostate cancer, renal carcinoma, SCLC, NSCLC and soft tissue tumor,and/or preventing postoperative recurrence thereof.

The present invention also contemplates pharmaceutical agents orcompositions that include or incorporate one or more peptides orpolynucleotides of the present invention formulated for the treatmentand/or prophylaxis of cancer, particularly a primary cancer, or theprevention of a postoperative recurrence thereof. Instead of or inaddition to the present peptides or polynucleotides, the presentpharmaceutical agents or compositions may include as active ingredientsAPCs or exosomes that present any of the present peptides.

The peptides or polynucleotides of the present invention may be used toinduce APCs which present on their surface a complex of an HLA antigenand the present peptide, for example, by contacting APCs derived from asubject with the peptide or introducing a polynucleotide encoding apeptide of this invention into APCs. Such APCs have high CTLinducibility against target peptides and are useful for cancerimmunotherapy. Accordingly, the present invention contemplates bothmethods for inducing APCs with CTL inducibility and the APCs obtained bysuch methods.

It is a further object of the present invention to provide methods forinducing CTLs, such methods including the step of co-culturingCD8-positive cells with APCs or exosomes presenting the peptide of thepresent invention on its surface or the step introducing apolynucleotide/polynucleotides encoding T cell receptor (TCR) subunitpolypeptides, wherein the TCR formed by such subunit polypeptides iscapable of binding to a complex of an HLA antigen and the presentpeptide on a cell surface. CTLs obtained by such methods are useful inthe treatment and prevention of cancers, examples of which include, butare not limited to, AML, CML, bladder cancer, breast cancer, cervicalcancer, colorectal cancer, esophagus cancer, liver cancer, osteosarcoma,prostate cancer, renal carcinoma, SCLC, NSCLC and soft tissue tumor.

Yet another object of the present invention is to provide isolated APCsthat present on the surface a complex of an HLA antigen and a peptide ofthe present invention. The present invention further provides isolatedCTLs that target peptides of the present invention. These APCs and CTLsmay be used for cancer immunotherapy.

It is yet another object of the present invention to provide methods forinducing an immune response against a cancer in a subject in needthereof, such methods including the step of administering to the subjecta composition or agent that include one or more peptides of the presentinvention, polynucleotides encoding the peptide of the presentinvention, or exosomes or APCs presenting the peptide of the presentinvention.

The applicability of the present invention extends to any of a number ofdiseases relating to or arising from TOMM34 overexpression, such ascancer, exemplary cancers including, but not limited to, AML, CML,bladder cancer, breast cancer, cervical cancer, colorectal cancer,esophagus cancer, liver cancer, osteosarcoma, prostate cancer, renalcarcinoma, SCLC, NSCLC and soft tissue tumor.

More specifically, the present invention provides the following [1] to[20]:

[1] An isolated peptide following (a) or (b):

(a) an isolated peptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1, 5 31 and 32;

(b) an isolated peptide comprising an amino acid sequence in which one,two, or several amino acid(s) are substituted, deleted, inserted oradded to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1, 5, 31 and 32 to yield a modified peptide that retains theability to bind to an HLA antigen and cytotoxic T lymphocyte (CTL)inducibility;

[2] The isolated peptide of [1], wherein the HLA antigen is HLA-A2;

[3] The isolated peptide of [1] or [2], wherein said peptide is anonapeptide or decapeptide;

[4] The peptide of any one of [1] to [3], having at least onesubstitution selected from the group consisting of:

(a) the second amino acid from N-terminus is or is modified to be anamino acid selected from the group consisting of leucine and methionine,and

(b) the C-terminal amino acid is or is modified to be an amino acidselected from the group consisting of valine and leucine;

[5] An isolated polynucleotide encoding the peptide of any one of [1] to[4];

[6] A composition for inducing CTL, wherein the composition comprisesone or more peptide(s) of any one of [1] to [4], or one or morepolynucleotide(s) of [5];

[7] A pharmaceutical composition comprising:

(a) one or more peptide(s) of any one of [1] to [4];

(b) one or more polynucleotides of [5];

(c) one or more APCs or exosomes that present a complex of the peptideof any one of [1] to [4] and an HLA antigen on their surface; or

(d) one or more CTLs that recognize a cell presenting a complex of thepeptide of any one of [1] to [4] and an HLA antigen on its surface,

in combination with pharmaceutically acceptable carrier,

formulated for a purpose selected from the group consisting of:

(i) treatment of an existing cancer,

(ii) prophylaxis of a cancer,

(iii) prevention of a postoperative recurrence of a cancer, and

(vi) combinations thereof;

[8] The pharmaceutical composition of [7] formulated for administrationto a subject whose HLA antigen is HLA-A2;

[9] A method for inducing an antigen-presenting cell (APC) with CTLinducibility, comprising a step selected from the group consisting of:

(a) contacting an APC with the peptide of any one of [1] to [4] invitro, ex vivo or in vivo, and

(b) introducing a polynucleotide encoding the peptide of any one of [1]to [4] into an APC.

[10] A method for inducing a CTL, comprising a step selected from thegroup consisting of:

(a) co-culturing a CD8-positive T cell with an APC that presents on itssurface a complex of an HLA antigen and the peptide of any one of [1] to[4],

(b) co-culturing a CD8-positive T cell with an exosome that presents onits surface a complex of an HLA antigen and a peptide of any one of [1]to [4], and

(c) introducing into a T cell a polynucleotide/polynucleotides encodingT cell receptor (TCR) subunit polypeptides, wherein the TCR formed bysaid TCR subunit polypeptides is capable of binding to a complex of anHLA antigen and the peptide of any one of [1] to [4] on a cell surface;

[11] An isolated APC that presents on its surface a complex of an HLAantigen and a peptide of any one of [1] to [4];

[12] The APC of [11], which is induced by a method for inducing anantigen-presenting cell (APC) with CTL inducibility, wherein the methodcomprises a step selected from the group consisting of:

(a) contacting an APC with the peptide of any one of [1] to [4] invitro, ex vivo or in vivo, and

(b) introducing a polynucleotide encoding the peptide of any one of [1]to [4] into an APC;

[13] An isolated CTL that targets any of the peptides of [1] to [4];

[14] The CTL of [13], which is induced by a method for inducing a CTL,wherein the method comprises a step selected from the group consistingof:

(a) co-culturing a CD8-positive T cell with an APC that presents on itssurface a complex of an HLA antigen and the peptide of any one of [1] to[4],

(b) co-culturing a CD8-positive T cell with an exosome that presents onits surface a complex of an HLA antigen and a peptide of any one of [1]to [4], and

(c) introducing into a T cell a polynucleotide/polynucleotides encodingT cell receptor (TCR) subunit polypeptides, wherein the TCR formed bysaid TCR subunit polypeptides is capable of binding to a complex of anHLA antigen and the peptide of any one of [1] to [4] on a cell surface;

[15] A method of inducing an immune response against cancer in asubject, comprising the step of administering to the subject a peptideof [1] to [4], an immunologically active fragment thereof, or apolynucleotide encoding the peptide or the fragment;

[16] A vector comprising a nucleotide sequence encoding the peptide ofany one of [1] to [4];

[17] A host cell transformed or transfected with an expression vector of[16];

[18] An exosome that presents a complex comprising the peptide of anyone of [1] to [4] and an HLA antigen;

[19] An antibody against the peptide of any one of [1] to [4], orimmunologically active fragment thereof; and

[20] A diagnostic kit comprising the peptide of any one of [1] to [4],the polynucleotide of [5] or antibody or immunologically active fragmentof [19].

Objects and features of the invention will become more fully apparentwhen the following detailed description is read in conjunction with theaccompanying figures and examples. It is to be understood that both theforegoing summary of the present invention and the following detaileddescription are of exemplified embodiments, and not restrictive of thepresent invention or other alternate embodiments of the presentinvention. In particular, while the invention is described herein withreference to a number of specific embodiments, it will be appreciatedthat the description is illustrative of the invention and is notconstructed as limiting of the invention. Various modifications andapplications may occur to those who are skilled in the art, withoutdeparting from the spirit and the scope of the invention, as describedby the appended claims. Likewise, other objects, features, benefits andadvantages of the present invention will be apparent from this summaryand certain embodiments described below, and will be readily apparent tothose skilled in the art. Such objects, features, benefits andadvantages will be apparent from the above in conjunction with theaccompanying examples, data, figures and all reasonable inferences to bedrawn therefrom, alone or with consideration of the referencesincorporated herein.

BRIEF DESCRIPTION OF DRAWINGS

Various aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the briefdescription of the figures and the detailed description of the presentinvention and its preferred embodiments that follows.

FIG. 1 is composed of a series of photographs, (a)-(e), depicting theresults of interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT)assay on CTLs that were induced with peptides derived from TOMM34. TheCTLs in well number #4 with TOMM34-A02-9-30 (SEQ ID NO: 1) (a), in #2with TOMM34-A02-9-220 (SEQ ID NO: 5) (b), in #4 with TOMM34-A02-10-30(SEQ ID NO: 31) (c) and in #2 with TOMM34-A02-10-220 (SEQ ID NO: 32) (d)showed potent IFN-gamma production as compared with the control,respectively. The square on the well of these pictures indicates thatthe cells from corresponding well were expanded to establish CTL lines.In contrast, as is the typical case of negative data, specific IFN-gammaproduction from the CTL stimulated with TOMM34-A02-10-143 (SEQ ID NO:21) (e) was not shown. In the figures, “+” indicates the IFN-gammaproduction against target cells pulsed with the appropriate peptide, and“−” indicates the IFN-gamma production against target cells not pulsedwith any peptides.

FIG. 2 is a line graph depicting the IFN-gamma production of the CTLline stimulated with TOMM34-A02-9-30 (SEQ ID NO: 1). The quantity ofIFN-gamma which CTL produced was measured by IFN-gamma enzyme-linkedimmunosorbent assay (ELISA). The results demonstrate that CTL lineestablished by stimulation with this peptide show potent TN-gammaproduction as compared with the control. In the figures, “+” indicatesthe IFN-gamma production against target cells pulsed with theappropriate peptide, and “−” indicates the IFN-gamma production againsttarget cells not pulsed with any peptides.

FIG. 3 is a line graph depicting the IFN-gamma production of the CTLclone established by limiting dilution from the CTL line stimulated withTOMM34-A02-9-30 (SEQ ID NO:1). The results demonstrate that the CTLclone established by stimulation with this peptide show potent IFN-gammaproduction as compared with the control. In the figure, “+” indicatesthe IFN-gamma production against target cells pulsed with theappropriate peptide and “−” indicates the IFN-gamma production againsttarget cells not pulsed with any peptides.

FIG. 4 is a line graph depicting the specific CTL activity againsttarget cells that express TOMM34 and HLA-A*0201. COS7 cells transfectedwith HLA-A′0201 or the full length TOMM34 gene were prepared as thecontrols. The CTL line established with TOMM34-A02-9-30 (SEQ TD NO:1)showed specific CTL activity against COS7 cells transfected with bothTOMM34 and HLA-A*0201 (black lozenge). On the other hand, no significantspecific CTL activity was detected against target cells expressingeither HLA-A*0201 (triangle) or TOMM34 (circle).

DESCRIPTION OF EMBODIMENTS

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. However, before the present materials and methods aredescribed, it should be understood that these descriptions are merelyillustrative and not intended to be limited. It should also beunderstood that the present invention is not limited to the particularsizes, shapes, dimensions, materials, methodologies, protocols, etc.described herein, as these may vary in accordance with routineexperimentation and optimization. It is also to be understood that theterminology used in the description is for the purpose of describing theparticular versions or embodiments only, and is not intended to limitthe scope of the present invention which will be limited only by theappended claims.

All publication, patent or patent application mentioned in thisspecification are specifically incorporated by reference herein in theirentirety. However, nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

I. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present invention belongs. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

The words “a”, “an”, and “the” as used herein mean “at least one” unlessotherwise specifically indicated.

The terms “isolated” and “purified” used in relation with a substance(e.g., peptide, antibody, polynucleotide, etc.) indicates that thesubstance is substantially free from at least one substance that mayelse be included in the natural source. Thus, an isolated or purifiedpeptide refers to peptide that are substantially free of cellularmaterial such as carbohydrate, lipid, or other contaminating proteinsfrom the cell or tissue source from which the peptide is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. The term “substantially free of cellularmaterial” includes preparations of a peptide in which the peptide isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, a peptide that issubstantially free of cellular material includes preparations ofpolypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight)of heterologous protein (also referred to herein as a “contaminatingprotein”). When the peptide is recombinantly produced, it is alsopreferably substantially free of culture medium, which includespreparations of peptide with culture medium less than about 20%, 10%, or5% of the volume of the peptide preparation. When the peptide isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, which includes preparations ofpeptide with chemical precursors or other chemicals involved in thesynthesis of the peptide less than about 30%, 20%, 10%, 5% (by dryweight) of the volume of the peptide preparation. That a particularpeptide preparation contains an isolated or purified peptide can beshown, for example, by the appearance of a single band following sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the proteinpreparation and Coomassie Brilliant Blue staining or the like of thegel. In a preferred embodiment, peptides and polynucleotides of thepresent invention are isolated or purified.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is a modified residue, or a non-naturally occurring residue,such as an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “oligopeptide” sometimes used in the present specification isused to refer to peptides of the present invention which are 20 residuesor fewer, typically 15 residues or fewer in length and is typicallycomposed of between about 8 and about 11 residues, often 9 or 10residues.

The term “amino acid” as used herein refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that similarly function to the naturally occurring amino acids.Amino acid may be either L-amino acids or D-amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose modified after translation in cells (e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine). The phrase “amino acidanalog” refers to compounds that have the same basic chemical structure(an alpha carbon bound to a hydrogen, a carboxy group, an amino group,and an R group) as a naturally occurring amino acid but have a modifiedR group or modified backbones (e.g., homoserine, norleucine, methionine,sulfoxide, methionine methyl sulfonium). The phrase “amino acid mimetic”refers to chemical compounds that have different structures but similarfunctions to general amino acids.

Amino acids may be referred to herein by their commonly known threeletter symbols or the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

The terms “gene”, “polynucleotides” and “nucleic acids” are usedinterchangeably herein and, unless otherwise specifically indicated aresimilarly to the amino acids referred to by their commonly acceptedsingle-letter codes.

The term “composition” as used herein is intended to encompass a productincluding the specified ingredients in the specified amounts, as well asany product that results, directly or indirectly, from combination ofthe specified ingredients in the specified amounts. Such term inrelation to “pharmaceutical composition”, is intended to encompass aproduct including the active ingredient(s), and any inert ingredient(s)that make up the carrier, as well as any product that results, directlyor indirectly, from combination, complexation or aggregation of any twoor more of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions or interactions of one ormore of the ingredients. Accordingly, in the context of the presentinvention, the term “pharmaceutical composition” refers to anycomposition made by admixing a compound or substance of the presentinvention and a pharmaceutically or physiologically acceptable carrier.The phrase “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier”, as used herein, means a pharmaceutically orphysiologically acceptable material, composition, substance, compound orvehicle, including, but are not limited to, a liquid or solid filler,diluent, excipient, solvent or encapsulating material.

The term “active ingredient” herein refers to a substance in acomposition that is biologically or physiologically active.Particularly, in the context of pharmaceutical composition, the term“active ingredient” refers to a substance that shows an objectivepharmacological effect. For example, in case of pharmaceuticalcompositions for use in the treatment or prevention of cancer, activeingredients in the compositions may lead to at least one biological orphysiologically action on cancer cells and/or tissues directly orindirectly. Preferably, such action may include reducing or inhibitingcancer cell growth, damaging or killing cancer cells and/or tissues, andso on. Typically, indirect effect of active ingredients is inductions ofCTLs recognizing or killing cancer cells. Before being formulated, the“active ingredient” may also be referred to as “bulk”, “drug substance”or “technical product”.

Unless otherwise defined, the term “cancer” refers to the cancersover-expressing TOMM34 gene, examples of which include, but are notlimited to, AML, CML, bladder cancer, breast cancer, cervical cancer,colorectal cancer, esophagus cancer, liver cancer, osteosarcoma,prostate cancer, renal carcinoma, SCLC, NSCLC and soft tissue tumor.

Unless otherwise defined, the terms “cytotoxic T lymphocyte”, “cytotoxicT cell” and “CTL” are used interchangeably herein and unless otherwisespecifically indicated, refer to a sub-group of T lymphocytes that arecapable of recognizing non-self cells (e.g., tumor cells, virus-infectedcells) and inducing the death of such cells.

Unless otherwise defined, the terms “HLA-A2” refers to the HLA-A2 typecontaining the subtypes, examples of which include, but are not limitedto, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205,HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0213, HLA-A*0216,HLA-A*0218, HLA-A*0219, HLA-A*0228 and HLA-A*0250.

Unless otherwise defined, the term “kit” as used herein, is used inreference to a combination of reagents and other materials. It iscontemplated herein that the kit may include microarray, chip, marker,and so on. It is not intended that the term “kit” be limited to aparticular combination of reagents and/or materials.

As used herein, in the context of a subject or patient, the phrase“subject's (or patient's) HLA antigen is HLA-A2” refers to that thesubject or patient homozygously or heterozygously possess HLA-A2 antigengene as the MHC (major histocompatibility complex) Class I molecule, andHLA-A2 antigen is expressed in cells of the subject or patient as an HLAantigen.

To the extent that the methods and compositions of the present inventionfind utility in the context of the “treatment” of cancer, a treatment isdeemed “efficacious” if it leads to clinical benefit such as, reductionin expression of TOMM34 gene, or a decrease in size, prevalence, ormetastatic potential of the cancer in the subject. When the treatment isapplied prophylactically, “efficacious” means that it retards orprevents cancers from forming or prevents or alleviates a clinicalsymptom of cancer. Efficaciousness is determined in association with anyknown method for diagnosing or treating the particular tumor type.

To the extent that the methods and compositions of the present inventionfind utility in the context of the “prevention” and “prophylaxis” ofcancer, such terms are interchangeably used herein to refer to anyactivity that reduces the burden of mortality or morbidity from disease.Prevention and prophylaxis can occur “at primary, secondary and tertiaryprevention levels.” While primary prevention and prophylaxis avoid thedevelopment of a disease, secondary and tertiary levels of preventionand prophylaxis encompass activities aimed at the prevention andprophylaxis of the progression of a disease and the emergence ofsymptoms as well as reducing the negative impact of an alreadyestablished disease by restoring function and reducing disease-relatedcomplications. Alternatively, prevention and prophylaxis can include awide range of prophylactic therapies aimed at alleviating the severityof the particular disorder, e.g. reducing the proliferation andmetastasis of tumors.

In the context of the present invention, the treatment and/orprophylaxis of cancer and/or the prevention of postoperative recurrencethereof include any of the following steps, such as the surgical removalof cancer cells, the inhibition of the growth of cancerous cells, theinvolution or regression of a tumor, the induction of remission andsuppression of occurrence of cancer, the tumor regression, and thereduction or inhibition of metastasis. Effective treatment and/or theprophylaxis of cancer decreases mortality and improves the prognosis ofindividuals having cancer, decreases the levels of tumor markers in theblood, and alleviates detectable symptoms accompanying cancer. Forexample, reduction or improvement of symptoms constitutes effectivelytreating and/or the prophylaxis include 10%, 20%, 30% or more reduction,or stable disease.

In the context of the present invention, the term “antibody” refers toimmunoglobulins and fragments thereof that are specifically reactive toa designated protein or peptide thereof. An antibody can include humanantibodies, primatized antibodies, chimeric antibodies, bispecificantibodies, humanized antibodies, antibodies fused to other proteins orradiolabels, and antibody fragments. Furthermore, an antibody herein isused in the broadest sense and specifically covers intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from at least two intact antibodies, andantibody fragments so long as they exhibit the desired biologicalactivity. An “antibody” indicates all classes (e.g., IgA, IgD, IgE, IgGand IgM).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

II. PEPTIDES

Peptides of the present invention described in detail below may bereferred to as TOMM34 peptide(s).

To demonstrate that peptides derived from TOMM34 function as an antigenrecognized by CTLs, peptides derived from TOMM34 (SEQ ID NO: 42) wereanalyzed to determine whether they were antigen epitopes restricted byHLA-A2 which are commonly encountered HLA alleles (Date Y et al., TissueAntigens 47: 93-101, 1996; Kondo A et al., J Immunol 155: 4307-12, 1995;Kubo R T et al., J Immunol 152: 3913-24, 1994). Candidates of HLA-A2binding peptides derived from TOMM34 were identified using theinformation on their binding affinities to HLA-A2. The followingcandidate peptides were identified;

TOMM34-A02-9-30 (SEQ ID NO: 1),

TOMM34-A02-9-77 (SEQ ID NO: 2),

TOMM34-A02-9-52 (SEQ ID NO: 3),

TOMM34-A02-9-110 (SEQ ID NO: 4),

TOMM34-A02-9-220 (SEQ TD NO: 5),

TOMM34-A02-9-230 (SEQ ID NO: 6),

TOMM34-A02-9-103 (SEQ ID NO: 7),

TOMM34-A02-9-80 (SEQ ID NO: 8),

TOMM34-A02-9-255 (SEQ ID NO: 9),

TOMM34-A02-9-23 (SEQ ID NO: 10),

TOMM34-A02-9-195 (SEQ ID NO: 11),

TOMM34-A02-9-111 (SEQ ID NO: 12),

TOMM34-A02-9-238 (SEQ TD NO: 13),

TOMM34-A02-9-1 (SEQ ID NO: 14),

TOMM34-A02-9-113 (SEQ ID NO: 15),

TOMM34-A02-9-253 (SEQ ID NO: 16),

TOMM34-A02-9-239 (SEQ ID NO: 17),

TOMM34-A02-9-144 (SEQ ID NO: 18),

TOMM34-A02-9-142 (SEQ ID NO: 19),

TOMM34-A02-9-279 (SEQ ID NO: 20),

TOMM34-A02-10-143 (SEQ ID NO: 21),

TOMM34-A02-10-97 (SEQ ID NO: 22),

TOMM34-A02-10-79 (SEQ ID NO: 23),

TOMM34-A02-10-237 (SEQ ID NO: 24),

TOMM34-A02-10-135 (SEQ ID NO: 25),

TOMM34-A02-10-219 (SEQ ID NO: 26),

TOMM34-A02-10-238 (SEQ ID NO: 27),

TOMM34-A02-10-127 (SEQ ID NO: 28),

TOMM34-A02-10-113 (SEQ ID NO: 29),

TOMM34-A02-10-241 (SEQ ID NO: 30),

TOMM34-A02-10-30 (SEQ ID NO: 31),

TOMM34-A02-10-220 (SEQ ID NO: 32),

TOMM34-A02-10-195 (SEQ ID NO: 33),

TOMM34-A02-10-112 (SEQ ID NO: 34),

TOMM34-A02-10-194 (SEQ ID NO: 35)

TOMM34-A02-10-299 (SEQ ID NO: 36)

TOMM34-A02-10-141 (SEQ ID NO: 37)

TOMM34-A02-10-160 (SEQ ID NO: 38)

TOMM34-A02-10-175 (SEQ ID NO: 39) and

TOMM34-A02-10-186 (SEQ ID NO: 40).

Moreover, after in vitro stimulation of T-cells by dendritic cells (DCs)loaded with these peptides, CTLs were successfully established usingeach of the following peptides;

TOMM34-A02-9-30 (SEQ ID NO: 1),

TOMM34-A02-9-220 (SEQ ID NO: 5),

TOMM34-A02-10-30 (SEQ ID NO: 31) and

TOMM34-A02-10-220 (SEQ ID NO: 32).

These established CTLs showed potent specific CTL activity againsttarget cells pulsed with respective peptides. The results hereindemonstrate that TOMM34 is an antigen recognized by CTL and that thepeptides tested are epitope peptides of TOMM34 restricted by HLA-A2.

Since the TOMM34 gene is over-expressed in cancer cells and tissues,including, but not limited to, those of AML, CML, bladder cancer, breastcancer, cervical cancer, colorectal cancer, esophagus cancer, livercancer, osteosarcoma, prostate cancer, renal carcinoma, SCLC, NSCLC andsoft tissue tumor but is not expressed in most normal organs, it is agood target for immunotherapy. Thus, the present invention providesnonapeptides (peptides composed of nine amino acid residues) anddecapeptides (peptides composed of ten amino acid residues) ofCTL-recognized epitopes from TOMM34. Alternatively, the presentinvention provides an isolated peptide that binds to an HLA antigen andinduces cytotoxic T lymphocytes (CTL), wherein the peptide has the aminoacid sequence of SEQ ID NO: 42 or is an immunologically active fragmentthereof. Specifically, the present invention provides peptidescomprising the amino acid sequence selected from among SEQ ID NOs: 1, 5,31 and 32. More specifically, in some embodiments, the present inventionprovides peptides consisting of the amino acid sequence selected fromamong SEQ ID NOs: 1, 5, 31 and 32.

Generally, software programs presently available, for example, on theInternet, such as those described in Parker K C et al., J Immunol 1994,152(1): 163-75, can be used to calculate the binding affinities betweenvarious peptides and HLA antigens in silico. Binding affinity with HLAantigens can be measured as described, for example, in Parker K C etal., J Immunol 1994, 152(1): 163-75; and Kuzushima K et al., Blood 2001,98(6): 1872-81, Larsen M V et al. BMC Bioinformatics. 2007; 8: 424, andBuus S et al. Tissue Antigens., 62:378-84, 2003. Methods for determiningbinding affinity are described, for example, in the Journal ofImmunological Methods, 1995, 185: 181-190 and Protein Science, 2000, 9:1838-1846. Therefore, one can readily utilize such software programs toselect those fragments derived from TOMM34 that have high bindingaffinity with HLA antigens. Accordingly, the present inventionencompasses peptides composed of any fragments derived from TOMM34 thathave high binding affinity with HLA antigens determined by such knownprograms. Furthermore, such peptides may include the full lengthsequence of TOMM34 (SEQ ID NO: 42).

The peptides of the present invention, particularly the nonapeptides anddecapeptides of the present invention, may be flanked with additionalamino acid residues so long as the peptide retains its CTL inducibility.The particular additional amino acid residues may be composed of anykind of amino acids so long as they do not impair the CTL inducibilityof the original peptide. Thus, the present invention encompassespeptides having binding affinity for HLA antigens, in particularincluding peptides derived from TOMM34. Such peptides are, for example,less than about 40 amino acids, often less than about 20 amino acids,usually less than about 15, 14, 13, 12, 11 or 10 amino acids.

Generally, it is known that modifications of one or more amino acids ina peptide do not influence the function of the peptide, or in some caseseven enhance the desired function of the original protein. In fact,modified peptides (i.e., peptides composed of an amino acid sequencemodified by substituting, deleting, inserting, or adding one, two orseveral amino acid residues to an original reference sequence) have beenknown to retain the biological activity of the original peptide (Mark etal., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, NucleicAcids Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl AcadSci USA 1982, 79: 6409-13). Thus, according to one embodiment of thepresent invention, the peptide having CTL inducibility of the presentinvention may be composed of the peptide consisting of the amino acidsequence selected from among SEQ ID NOs: 1, 5, 31 and 32, in which one,two or even more amino acids are added, deleted, inserted and/orsubstituted.

One of skill in the art will recognize that individual modifications(i.e., deletions, insertions, additions or substitutions) to an aminoacid sequence which alters a single amino acid or a small percentage ofthe overall amino acid sequence results in the conservation of theproperties of the original amino acid side-chain; it is thus referred toas “conservative substitution” or “conservative modification”, whereinthe alteration of a protein results in a protein with similar functions.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Examples of properties of amino acidside chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and sidechains having the following functional groups or characteristics incommon: an aliphatic side-chain (G, A, V, L, T, P); a hydroxyl groupcontaining side-chain (S, T, Y); a sulfur atom containing side-chain (C,M); a carboxylic acid and amide containing side-chain (D, N, E, Q); abase containing side-chain (R, K, H); and an aromatic containingside-chain (H, F, Y, W). In addition, the following eight groups eachcontain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (T), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).

Such conservatively modified peptides are also considered to be peptidesof the present invention. However, the peptide of the present inventionis not restricted thereto and may include non-conservativemodifications, so long as the resulting modified peptide retains the CTLinducibility of the original unmodified peptide. Furthermore, themodified peptides do not exclude CTL inducible peptides of polymorphicvariants, interspecies homologues, and alleles of TOMM34.

Amino acid residues may be inserted, substituted or added to thepeptides of the present invention or, alternatively, amino acid residuesmay be deleted therefrom to achieve a higher binding affinity. To retainthe requisite CTL inducibility, one preferably modifies (i.e., deletes,inserts, adds or substitutes) a small number (for example, 1, 2 orseveral) or a small percentage of amino acids. Herein, the term“several” means 5 or fewer amino acids, for example, 3 or fewer. Thepercentage of amino acids to be modified may be 20% or less, forexample, 15% or less, for example 10% or less, for example 1 to 5%.

Moreover, the peptides may be substituted or added by such of the aminoacid residues to achieve a higher binding affinity. When used inimmunotherapy, the present peptides are presented on the surface of acell or exosome as a complex with an HLA antigen. In addition topeptides that are naturally displayed, since the regularity of thesequences of peptides displayed by binding to HLA antigens is alreadyknown (J Immunol 1994, 152: 3913; Immunogenetics 1995, 41: 178; JImmunol 1994, 155: 4307), modifications based on such regularity may beintroduced into the immunogenic peptides of the present invention.

For example, peptides possessing high HLA-A2 binding affinity tend tohave the second amino acid from the N-terminus substituted with leucineor methionine. Likewise, peptides in which the C-terminal is substitutedwith valine or leucine can also be favorably used. Accordingly, peptideshaving the amino acid sequences selected from among SEQ ID NOs: 1, 5, 31and 32 in which the second amino acid from the N-terminus of the aminoacid sequence of said SEQ ID NO is substituted with leucine ormethionine, and/or the C-terminus of the amino acid sequence of said SEQID NO is substituted with valine or leucine are encompassed by thepresent invention. Substitutions may be introduced not only at theterminal amino acids but also at the position of potential T cellreceptor (TCR) recognition of peptides. Several studies havedemonstrated that a peptide with amino acid substitutions may be equalto or better than the original, for example CAP1, p53₍₂₆₄₋₂₇₂₎,Her-2/neu₍₃₆₉₋₃₇₇₎, or gp100₍₂₀₉₋₂₁₇₎ (Zaremba et al. Cancer Res. 57,4570-4577, 1997, T. K. Hoffmann et al. J Immunol. (2002);168(3):1338-47, S. O. Dionne et al. Cancer Immunol immunother. (2003)52: 199-206 and S. O. Dionne et al. Cancer Immunology, Immunotherapy(2004) 53, 307-314).

The present invention also contemplates the addition of one, two orseveral amino acids to the N and/or C-terminus of the present peptides.Such modified peptides with high HLA antigen binding affinity andretained CTL inducibility are also included in the present invention.

For example, the present invention provides an isolated peptide of lessthan 14, 13, 12, 11, or 10 amino acids in length which binds an HLAantigen, has CTL inducibility, and comprises the amino acid sequenceselected from among:

(i) an amino acid sequence is selected from among SEQ ID NOs: 1 and 5;

(ii) an amino acid sequence in which one, two or several amino acid(s)are modified in the amino acid sequence selected from among SEQ ID NOs:1 and 5, and

(iii) the amino acid sequence of (ii), wherein the amino acid sequencehas one or both of the following characteristics:

(a) the second amino acid from the N-terminus of said SEQ ID NO isselected from among leucine and methionine; and

(b) the C-terminal amino acid of said SEQ ID NO is selected from amongvaline and leucine.

Moreover, the present invention also provides an isolated peptide ofless than 15, 14, 13, 12, or 11 amino acids in length which binds an HLAantigen, has CTL inducibility, and comprises the amino acid sequenceselected from among:

(i′) an amino acid sequence is selected from among SEQ ID NOs: 31 and32;

(ii′) an amino acid sequence in which one, two or several amino acid(s)are modified in the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 31 and 32, and

(iii′) the amino acid sequence of (ii′), wherein the amino acid sequencehas one or both of the following characteristics:

(a) the second amino acid from the N-terminus of said SEQ TD NO isselected from among leucine and methionine; and

(b) the C-terminal amino acid of said SEQ ID NO is selected from amongvaline and leucine.

When these peptides are contacted with APCs, those peptides bind withHLA antigens on APCs to be presented on APCs as complexes with HLAantigens. Alternatively, those peptides are introduced into APCs andprocessed to fragments consisting of an amino acid sequence selectedfrom among (i)-(iii) and (i′)-(iii′) in APCs to be presented on APCs ascomplexes with HLA antigens. Consequently, CTLs specific to suchpeptides are induced.

However, when the peptide sequence is identical to a portion of theamino acid sequence of an endogenous or exogenous protein having adifferent function, side effects such as autoimmune disorders orallergic symptoms against specific substances may be induced. Therefore,one can perform homology searches using available databases to avoidsituations in which the sequence of the peptide matches the amino acidsequence of another protein. When it becomes clear from the homologysearches that there exists not even a peptide with 1 or 2 amino acidsdifference to the objective peptide, the objective peptide may bemodified in order to increase its binding affinity with HLA antigens,and/or increase its CTL inducibility without any danger of such sideeffects.

Although peptides having high binding affinity to the HLA antigens asdescribed above are expected to be highly effective, the candidatepeptides, which are selected according to the presence of high bindingaffinity as an indicator, are further examined for the presence of CTLinducibility. Herein, the phrase “CTL inducibility” indicates theability of the peptide to induce CTLs when presented onantigen-presenting cells (APCs). Further, “CTL inducibility” includesthe ability of the peptide to induce CTL activation, CTL proliferation,promote CTL lysis of target cells, and to increase CTL IFN-gammaproduction.

Confirmation of CTL inducibility may be accomplished by inducing APCscarrying human MHC antigens (for example, B-lymphocytes, macrophages,and dendritic cells (DCs)), or more specifically DCs derived from humanperipheral blood mononuclear leukocytes, and after stimulation with thepeptides, mixing with CD8-positive T cells, and then measuring theIFN-gamma produced and released by CTL against the target cells. As thereaction system, transgenic animals that have been produced to express ahuman HLA antigen (for example, those described in BenMohamed L,Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond D J, HumImmunol 2000, 61(8): 764-79, Related Articles, Books, Linkout Inductionof CTL response by a minimal epitope vaccine in HLA-A*0201/DR1transgenic mice: dependence on HLA class II restricted T(H) response)can be used. For example, the target cells may be radiolabeled with ⁵¹Crand such, and cytotoxic activity may be calculated from radioactivityreleased from the target cells. Alternatively, it may be examined bymeasuring IFN-gamma produced and released by CTL in the presence of APCsthat carry immobilized peptides, and visualizing the inhibition zone onthe media using anti-IFN-gamma monoclonal antibodies.

By examining the CTL inducibility of the peptides as described above, itwas discovered that nonapeptides or decapeptides having an amino acidsequence selected from among SEQ ID NOs: 1, 5, 31 and 32 showedparticularly high CTL inducibility as well as high binding affinity toan HLA antigen. Thus, these peptides are exemplified as preferredembodiments of the present invention.

Furthermore, homology analyses demonstrated that such peptides do nothave significant homology with peptides derived from any other knownhuman gene products. Accordingly, the possibility of unknown orundesired immune responses arising when used for immunotherapy may belowered. Therefore, also from this aspect, these peptides find use foreliciting immunity in cancer patients against TOMM34. Thus, thepreferred peptides of the present invention are those peptidesconsisting of the amino acid sequence selected from the group consistingof SEQ ID NOs: 1, 5, 31 and 32.

In addition to modifications of the present peptides, discussed above,the peptides of the present invention may also be linked to otherpeptides, so long as they retain the CTL inducibility, and morepreferably also retains the requisite HLA binding. Examples of suitable“other” peptides include: the peptides of the present invention or theCTL inducible peptides derived from other TAAs. The linkers between thepeptides are well known in the art, for example, AAY (P. M. Daftarian etal., J Trans Med 2007, 5:26), AAA, NKRK (R. P. M. Sutmuller et al., JImmunol. 2000, 165: 7308-7315) or K (S. Ota et al., Can Res. 62,1471-1476, K. S. Kawamura et al., J Immunol. 2002, 168: 5709-5715).

For example, non-TOMM34 tumor associated antigen peptides also can beused substantially simultaneously to increase the immune response viaHLA class I and/or class II. It is well established that cancer cellscan express more than one tumor associated gene. Thus, it is within thescope of routine experimentation for one of ordinary skill in the art todetermine whether a particular subject expresses additional tumorassociated genes, and then include HLA class I and/or HLA class IIbinding peptides derived from expression products of such genes inTOMM34 compositions or vaccines.

Examples of HLA class I and HLA class II binding peptides are known tothose of ordinary skill in the art (for example, see Coulie, Stem Cells13:393-403, 1995), and thus can be used in the invention in a likemanner as those disclosed herein. Thus, one of ordinary skill in the artcan readily prepare polypeptides including one or more TOMM34 peptidesand one or more of the non-TOMM34 peptides, or nucleic acids encodingsuch polypeptides, using standard procedures of molecular biology.

The above described peptides are referred to herein as “polytopes”,i.e., groups of two or more potentially immunogenic or immune responsestimulating peptides which can be joined together in variousarrangements (e.g., concatenated, overlapping). The polytope (or nucleicacid encoding the polytope) can be administered in a standardimmunization protocol, e.g., to animals, to test the effectiveness ofthe polytope in stimulating, enhancing and/or provoking an immuneresponse.

The peptides can be joined together directly or via the use of flankingsequences to form polytopes, and the use of polytopes as vaccines iswell known in the art (see, e.g., Thomson et al., Proc. Natl. Acad. SciUSA 92(13):5845-5849, 1995; Gilbert et al., Nature Biotechnol.15(12):1280-1284, 1997; Thomson et al., J Immunol. 157(2):822-826, 1996;Tarn et al., J Exp. Med. 171(1):299-306, 1990). Polytopes containingvarious numbers and combinations of epitopes can be prepared and testedfor recognition by CTLs and for efficacy in increasing an immuneresponse.

The peptides of the present invention may be further linked to othersubstances, so long as they retain the CTL inducibility of the originalpeptide. Examples of suitable substances may include: peptides, lipids,sugar and sugar chains, acetyl groups, natural and synthetic polymers,etc. The peptides may contain modifications such as glycosylation, sidechain oxidation, or phosphorylation; so long as the modifications do notdestroy the biological activity of the peptides as described herein.These kinds of modifications may be performed to confer additionalfunctions (e.g., targeting function, and delivery function) or tostabilize the polypeptide.

For example, to increase the in vivo stability of a polypeptide, it isknown in the art to introduce D-amino acids, amino acid mimetics orunnatural amino acids; this concept may also be adopted for the presentpolypeptides. The stability of a polypeptide may be assayed in a numberof ways. For instance, peptidases and various biological media, such ashuman plasma and serum, can be used to test stability (see, e.g.,Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).

Moreover, as noted above, among the modified peptides that aresubstituted, deleted, inserted or added by one, two or several aminoacid residues, those having same or higher activity as compared tooriginal peptides can be screened for or selected. The presentinvention, therefore, also provides the method of screening for orselecting modified peptides having same or higher activity as comparedto originals. For example, the method may include steps of:

a: substituting, deleting, inserting, or adding at least one amino acidresidue of a peptide of the present invention,

b: determining the activity of said peptide, and

c: selecting the peptide having same or higher activity as compared tothe original.

Herein, said activity may include MHC binding activity, APC or CTLinducibility and cytotoxic activity.

When the peptides of the present intention include a cysteine residue,the peptides tend to form dimers via a disulfide bond between SH groupsof the cysteine residues. Therefore, dimers of the peptide of thepresent invention are also included in the peptides of the presentinvention.

III. PREPARATION OF TOMM34 PEPTIDES

The peptides of the present invention may be prepared using well knowntechniques. For example, the peptides may be prepared synthetically, byrecombinant DNA technology or chemical synthesis. The peptides of thepresent invention may be synthesized individually or as longerpolypeptides including two or more peptides. The peptides may beisolated, i.e., purified or isolated substantially free of othernaturally occurring host cell proteins and fragments thereof, or anyother chemical substances.

The peptides of the present invention may contain modifications, such asglycosylation, side chain oxidation, or phosphorylation provided suchmodifications do not destroy the biological activity of the originalpeptide. Other illustrative modifications include incorporation ofD-amino acids or other amino acid mimetics that may be used, forexample, to increase the serum half life of the peptides.

A peptide of the present invention may be obtained through chemicalsynthesis based on the selected amino acid sequence. For example,conventional peptide synthesis methods that may be adopted for thesynthesis include:

(i) Peptide Synthesis, Interscience, New York, 1966;

(ii) The Proteins, Vol. 2, Academic Press, New York, 1976;

(iii) Peptide Synthesis (in Japanese), Maruzen Co., 1975;

(iv) Basics and Experiment of Peptide Synthesis (in Japanese), MaruzenCo., 1985;

(v) Development of Pharmaceuticals (second volume) (in Japanese), Vol.14 (peptide synthesis), Hirokawa, 1991;

(vi) WO99/67288; and

(vii) Barmy G. & Merrifield R. B., Peptides Vol. 2, “Solid Phase PeptideSynthesis”, Academic Press, New York, 1980, 100-118.

Alternatively, the present peptides may be obtained adapting any knowngenetic engineering methods for producing peptides (e.g., Morrison J, JBacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods inEnzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, asuitable vector harboring a polynucleotide encoding the objectivepeptide in an expressible form (e.g., downstream of a regulatorysequence corresponding to a promoter sequence) is prepared andtransformed into a suitable host cell. Such vectors and host cells arealso provided by the present invention. The host cell is then culturedto produce the peptide of interest. The peptide may also be produced invitro adopting an in vitro translation system.

IV. POLYNUCLEOTIDES

The present invention provides polynucleotide that encode any of theaforementioned peptides of the present invention. These includepolynucleotides derived from the natural occurring TOMM34 gene (forexample, SEQ ID NO: 42 (GenBank Accession No. NM_006809)) and thosehaving a conservatively modified nucleotide sequences thereof. Herein,the phrase “conservatively modified nucleotide sequence” refers tosequences which encode identical or essentially identical amino acidsequences. Due to the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG, and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon may be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a peptide also describes every possible silent variationof the nucleic acid. One of ordinary skill in the art will recognizethat each codon in a nucleic acid (except AUG, which is ordinarily theonly codon for methionine, and TGG, which is ordinarily the only codonfor tryptophan) may be modified to yield a functionally identicalmolecule. Accordingly, each silent variation of a nucleic acid thatencodes a peptide is implicitly described in each disclosed sequence.

The polynucleotide of the present invention may be composed of DNA, RNA,and derivatives thereof. As is well known in the art, a DNA molecule iscomposed of bases such as the naturally occurring bases A, T, C, and G,and T is replaced by U in an RNA. One of skill will recognize thatnon-naturally occurring bases be included in polynucleotides, as well.

The polynucleotide of the present invention may encode multiple peptidesof the present invention with or without intervening amino acidsequences. For example, the intervening amino acid sequence may providea cleavage site (e.g., enzyme recognition sequence) of thepolynucleotide or the translated peptides. Furthermore, thepolynucleotide may include any additional sequences to the codingsequence encoding the peptide of the present invention. For example, thepolynucleotide may be a recombinant polynucleotide that includesregulatory sequences required for the expression of the peptide or maybe an expression vector (plasmid) with marker genes and such. Ingeneral, such recombinant polynucleotides may be prepared by themanipulation of polynucleotides through conventional recombinanttechniques using, for example, polymerases and endonucleases.

Both recombinant and chemical synthesis techniques may be used toproduce the polynucleotides of the present invention. For example, apolynucleotide may be produced by insertion into an appropriate vector,which may be expressed when transfected into a competent cell.Alternatively, a polynucleotide may be amplified using PCR techniques orexpression in suitable hosts (see, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,1989). Alternatively, a polynucleotide may be synthesized using thesolid phase techniques, as described in Beaucage S L & Iyer R P,Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.

V. EXOSOMES

The present invention further provides intracellular vesicles, referredto as exosomes, that present complexes formed between the peptides ofthis invention and HLA antigens on their surface. Exosomes may beprepared, for example, using the methods detailed in Japanese PatentApplication Kohyo Publications Nos. Hei 11-510507 and WO99/03499, andmay be prepared using APCs obtained from patients who are subject totreatment and/or prevention. The exosomes of this invention may beinoculated as vaccines, similarly to the peptides of this invention.

The type of HLA antigens included in the complexes must match that ofthe subject requiring treatment and/or prevention. For example, for inthe Japanese population, HLA-A2, particularly HLA-A*0201 and HLA-A*0206,are quite prevalent and therefore would be appropriate for treatment ofJapanese patients. The use of HLA-A2 type that is highly expressed amongthe Japanese and Caucasian populations is favorable for obtainingeffective results, and subtypes such as HLA-A*0201 and HLA-A*0206 finduse. Typically, in the clinic, the type of HLA antigen of the patientrequiring treatment is investigated in advance, which enablesappropriate selection of peptides having high levels of binding affinityto this antigen, or having CTL inducibility by antigen presentation.Furthermore, in order to obtain peptides showing high binding affinityand CTL inducibility, substitution, deletion, insertion or addition ofone, two, or several amino acids may be performed based on the aminoacid sequence of the naturally occurring TOMM34 partial peptide.

When the exosome of the present invention possess HLA-A2 type as an HLAantigen, the peptides including the amino acid sequence selected fromamong SEQ ID NOs: 1, 5, 31 and 32 have particular utility.

VI. ANTIGEN-PRESENTING CELLS (APCS)

The present invention also provides isolated antigen-presenting cells(APCs) that present complexes formed between HLA antigens and thepeptides of this invention on its surface. The APCs may be derived frompatients who are subject to treatment and/or prevention, and may beadministered as vaccines by themselves or in combination with otherdrugs including the peptides of this invention, exosomes, or CTLs.

The APCs are not limited to a particular kind of cells. Examples of APCsinclude, but are not limited to, dendritic cells (DCs), Langerhanscells, macrophages, B cells, and activated T cells, which are known topresent proteinaceous antigens on their cell surface so as to berecognized by lymphocytes. Since DCs are a representative APCs havingthe strongest CTL inducing action among APCs, DCs find use as the APCsof the present invention.

For example, the APCs of the present invention may be obtained byinducing DCs from peripheral blood monocytes and then contacting(stimulating) them with the peptides of this invention in vitro, ex vivoor in vivo. When the peptides of this invention are administered to thesubjects, APCs that present the peptides of this invention are inducedin the body of the subject. The phrase “inducing an APC” includescontacting (stimulating) a cell with the peptides of the presentinvention, or introducing a polynucleotide encoding the peptides of thepresent invention into a cell to present a complex formed between an HLAantigen and the peptides of the present invention on cell's surface.Therefore, the APCs of this invention may be obtained by collecting theAPCs from the subject after administering the peptides of this inventionto the subject. Alternatively, the APCs of this invention may beobtained by contacting APCs collected from a subject with the peptide ofthis invention.

The APCs of the present invention may be administered to a subject forinducing immune response against cancer in the subject by themselves orin combination with other drugs including the peptides, exosomes or CTLsof this invention. For example, the ex vivo administration may includesteps of:

a: collecting APCs from a first subject,

b: contacting with the APCs of step a, with the peptide, and

c: administering the APCs of step b to a second subject.

The first subject and the second subject may be the same individual, ormay be different individuals. The APCs obtained by step b may serve as avaccine for the treatment and/or prevention of a cancer, examples ofwhich include, but are not limited to, AML, CML, bladder cancer, breastcancer, cervical cancer, colorectal cancer, esophagus cancer, livercancer, osteosarcoma, prostate cancer, renal carcinoma, SCLC, NSCLC andsoft tissue tumor.

In the context of the present invention, one may utilize the peptides ofthe present invention for manufacturing a pharmaceutical composition oragent capable of inducing antigen-presenting cells. A method or processfor manufacturing a pharmaceutical composition or agent for inducingantigen-presenting cells is provided herein and preferably includes thestep of admixing or formulating the peptide of the invention with apharmaceutically acceptable carrier.

The present invention also provides for the use of the peptides of thepresent invention for inducing antigen-presenting cells.

According to an aspect of the present invention, the APCs of the presentinvention have a high level of CTL inducibility. In the phrase “highlevel of CTL inducibility”, the high level means that CTL inducibilityis relatively high as compared to the level of that detected in APCscontacted with no peptide. Such APCs having a high level of CTLinducibility may be prepared by a method which includes the step oftransferring a polynucleotide encoding the peptide of this invention toAPCs in vitro as well as the method mentioned above. The introducedpolynucleotides may be in the form of DNAs or RNAs. Examples of methodsfor introduction include, without particular limitations, variousmethods conventionally performed in this field, such as lipofection,electroporation, and calcium phosphate method may be used. Morespecifically, it may be performed as described in Cancer Res 1996, 56:5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72;Published Japanese Translation of International Publication No.2000-509281. By transferring the gene encoding the peptide of thepresent invention into APCs, the gene undergoes transcription,translation, and such in the cell, and then the obtained protein isprocessed by MHC Class I or Class II, and proceeds through apresentation pathway to present the peptides of the present invention.

Alternatively, the APCs of the present invention can be prepared by amethod which includes the step of contacting APCs with the peptide ofthe present invention. In some embodiments, the APCs of the presentinvention present complexes of HLA-A2 antigen and the peptide of thepresent invention on their surface.

VII. CYTOTOXIC T LYMPHOCYTES (CTLS)

A CTL induced against any of the peptides of the present inventionstrengthens the immune response targeting cancer cells in vivo and thusmay be used as vaccines similar to the peptides per se. Thus, thepresent invention provides isolated CTLs that are specifically inducedor activated by any of the present peptides.

Such CTLs may be obtained by (1) administering the peptide(s) of thepresent invention to a subject, (2) contacting (stimulating)subject-derived APCs and CD8-positive T cells, or peripheral bloodmononuclear leukocytes in vitro with the peptide(s) of the presentinvention, (3) contacting CD8-positive T cells or peripheral bloodmononuclear leukocytes in vitro with the APCs or exosomes presenting acomplex of an HLA antigen and the peptide of the present invention onits surface or (4) introducing a polynucleotide/polynucleotides encodingT cell receptor (TCR) subunits, wherein the TCR formed by such TCRsubunits is capable of binding to a complex of an HLA antigen and thepeptide of this invention on a cell surface. Such APCs or exosomes forthe method of (3) can be prepared by the methods described above.Details of the method of (4) is described bellow in section “VIII. Tcell receptor (TCR)”.

The CTLs of the present invention may be derived from patients who aresubject to treatment and/or prevention, and may be administered bythemselves or in combination with other drugs including the peptides ofthis invention or exosomes for the purpose of regulating effects. Theobtained CTLs act specifically against target cells presenting thepeptides of this invention, for example, the same peptides used forinduction. The target cells may be cells that endogenously expressTOMM34, such as cancer cells, or cells that are transfected with theTOMM34 gene; and cells that present a peptide of this invention on thecell surface due to stimulation by the peptide may also serve as targetsof activated CTL attack.

In some embodiments, the CTLs of the present invention are CTLs thatrecognize cells presenting complexes of an HLA-A2 antigen and thepeptide of the present invention on their surface. In the context of theCTL, the phrase “recognize a cell” refers to binding a complex of anHLA-A2 antigen and the peptide of the present invention on the cellsurface via its TCR and showing specific cytotoxic activity against thecell. Herein, “specific cytotoxic activity” refers to showing cytotoxicactivity against the cell presenting a complex of an HLA-A2 antigen andthe peptide of the present invention but not other cells.

VIII. T CELL RECEPTOR (TCR)

The present invention also provides a composition including apolynucleotide/polynucleotides encoding polypeptides that are capable offorming a subunit of a T cell receptor (TCR), and methods of using thesame. Such TCR subunits have the ability to form TCRs that conferspecificity against tumor cells expressing TOMM34 to T cells. By usingthe known methods in the art, the polynucleotide/polynucleotidesencoding each of alpha- and beta-chains of the TCR subunits of the CTLinduced with the peptides of the present invention can be identified(WO2007/032255 and Morgan et al., J Immunol, 171, 3288 (2003)). Forexample, the PCR method is preferred to analyze the TCR. The PCR primersfor the analysis can be, for example, 5′-R primers(5′-gtctaccaggcattcgcttcat-3′) (SEQ ID NO: 43) as 5′ side primers and3-TRa-C primers (5′-tcagctggaccacagccgcagcgt-3′) (SEQ ID NO: 44)specific to TCR alpha chain C region, 3-TRb-C1 primers(5′-tcagaaatcctttctcttgac-3′) (SEQ ID NO: 45) specific to TCR beta chainC1 region or 3-TRbeta-C2 primers (5′-ctagcctctggaatcctttctctt-3′) (SEQID NO: 46) specific to TCR beta chain C2 region as 3′ side primers, butnot limited thereto. The derivative TCRs can bind target cellspresenting the TOMM34 peptide of the present invention with highavidity, and optionally mediate efficient killing of target cellspresenting the TOMM34 peptide of the present invention in vivo and invitro.

The polynucleotide/polynucleotides encoding the TCR subunits (i.e., thepolynucleotide encoding both of the TCR subunits or polynucleotidesencoding each of the TCR subunits) may be incorporated into suitablevectors, e.g., retroviral vectors. These vectors are well known in theart. The polynucleotide or the vectors including them usefully may betransferred into a T cell (e.g., CD8-positive T cell), for example, a Tcell from a patient. Advantageously, the present invention provides anoff-the-shelf composition allowing rapid modification of a patient's ownT cells (or those of another mammal) to rapidly and easily producemodified T cells having excellent cancer cell killing properties.

The specific TCR against the peptide of the present invention is areceptor capable of specifically recognizing a complex of a peptide ofthe present invention and HLA antigen, giving a T cell specific activityagainst a target cell presenting a complex of the peptide of the presentinvention and an HLA antigen when the TCR is expressed on the surface ofthe T cell. It can be confirmed by any known methods that CTLs preparedby introducing the polypeptide(s) encoding such TCR subunits can bespecifically recognize such target cells. Preferred examples of suchmethods include, for example, tetramer analysis using HLA molecule andthe peptide of the present invention, and ELISPOT assay. By ELISPOTassay, it can be confirmed that CTL prepared by the method describedabove can specifically recognizes the target cells and that the signalsgenerated by such recognition can be transmitted intracellularly.Furthermore, it can be also confirmed by known methods that CTLsprepared by the method described above have specific cytotoxic activityagainst the target cells. Examples of such methods include, for example,chromium release assay using cells expressing both of HLA-A2 antigen andTOMM34.

In one aspect, the present invention provides CTLs that are prepared bytransduction with the polynucleotide/polynucleotides encoding the TCRsubunits polypeptides (i.e., the polynucleotide encoding both of the TCRsubunits or polynucleotides encoding each of the TCR subunits), whereinthe TCR formed by such TCR subunits can bind to a complex of the TOMM34peptide having an amino acid sequence selected from among SEQ ID NOs: 1,5, 31 and 32 and HLA-A2 antigen on a cell surface.

The transduced CTLs are capable of homing to cancer cells in vivo, andmay be expanded by well known culturing methods in vitro (e.g., Kawakamiet al., J Immunol., 142, 3452-3461 (1989)). The CTLs of the presentinvention may be used to form an immunogenic composition useful ineither or both of the treatment and the prevention of cancer in apatient in need of therapy or protection (WO2006/031221).

IX. PHARMACEUTICAL AGENTS OR COMPOSITIONS

Since TOMM34 expression is specifically elevated in cancer, examples ofwhich include, but not limited to, AML, CML, bladder cancer, breastcancer, cervical cancer, colorectal cancer, esophagus cancer, livercancer, osteosarcoma, prostate cancer, renal carcinoma, SCLC, NSCLC andsoft tissue tumor as compared with normal tissue, the peptides of orpolynucleotides of the present invention may be used for the treatmentand/or prophylaxis of cancer, and/or the prevention of a postoperativerecurrence thereof. Thus, the present invention provides apharmaceutical agent or composition formulated for the treatment and/orprophylaxis of cancer, and/or for the prevention of a postoperativerecurrence thereof, such composition including as an active ingredientone or more of the peptides, or polynucleotides of this invention as anactive ingredient. Alternatively, the present peptides may be expressedon the surface of any of the foregoing exosomes or cells, such as APCsfor the use as pharmaceutical agents or compositions. In addition, theaforementioned CTLs which target any of the peptides of the presentinvention may also be used as the active ingredient of the presentpharmaceutical agents or compositions.

The pharmaceutical compositions of the present invention also find useas a vaccine. In the context of the present invention, the phrase“vaccine” (also referred to as an “immunogenic composition”) refers to acomposition that has the function to improve, enhance, and/or induceanti-tumor immunity upon inoculation into animals.

The pharmaceutical compositions of the present invention can be used totreat and/or prevent cancers, and/or prevention of postoperativerecurrence thereof in subjects or patients including human and any othermammal including, but not limited to, mouse, rat, guinea-pig, rabbit,cat, dog, sheep, goat, pig, cattle, horse, monkey, baboon, andchimpanzee, particularly a commercially important animal or adomesticated animal.

In another embodiment, the present invention also provides the use of anactive ingredient in manufacturing a pharmaceutical composition or agentfor treating cancer or tumor, said active ingredient selected fromamong:

(a) a peptide of the present invention;

(b) a nucleic acid encoding such a peptide as disclosed herein in anexpressible form;

(c) an APC or an exosome presenting a peptide of the present inventionon its surface; and

(d) a cytotoxic T cell of the present invention.

Alternatively, the present invention further provides an activeingredient for use in the treatment and/or prevention of cancers ortumors, said active ingredient selected from among:

(a) a peptide of the present invention;

(b) a nucleic acid encoding such a peptide as disclosed herein in anexpressible form;

(c) an APC or an exosome presenting a peptide of the present inventionon its surface; and

(d) a cytotoxic T cell of the present invention.

Alternatively, the present invention further provides a method orprocess for manufacturing a pharmaceutical composition or agent fortreating or preventing cancer or tumor, wherein the method or processincludes the step of formulating a pharmaceutically or physiologicallyacceptable carrier with an active ingredient selected from among:

(a) a peptide of the present invention;

(b) a nucleic acid encoding such a peptide as disclosed herein in anexpressible form;

(c) an APC or an exosome presenting a peptide of the present inventionon its surface; and

(d) a cytotoxic T cell of the present invention.

In another embodiment, the present invention also provides a method orprocess for manufacturing a pharmaceutical composition or agent fortreating or preventing cancer or tumor, wherein the method or processincludes the steps of admixing an active ingredient with apharmaceutically or physiologically acceptable carrier, wherein theactive ingredient is selected from among:

(a) a peptide of the present invention;

(b) a nucleic acid encoding such a peptide as disclosed herein in anexpressible form;

(c) an APC or an exosome presenting a peptide of the present inventionon its surface; and

(d) a cytotoxic T cell of the present invention.

The pharmaceutical agents or compositions of the present invention maybe used to treat and/or prevent cancer, and/or to prevent apostoperative recurrence thereof in subjects or patients including humanand any other mammal including, but not limited to, mouse, rat,guinea-pig, rabbit, cat, dog, sheep, goat, pig, cattle, horse, monkey,baboon, and chimpanzee, particularly a commercially important animal ora domesticated animal.

According to the present invention, peptides including the amino acidsequence selected from among SEQ ID NO: 1, 5, 31 and 32 have been foundto be HLA-A2 restricted epitope peptides or the candidates that mayinduce potent and specific immune response. Therefore, the presentpharmaceutical agents or compositions which include any of thesepeptides with the amino acid sequences of SEQ ID NOs: 1, 5, 31 and 32are particularly suited for the administration to subjects whose HLAantigen is HLA-A2. The same applies to pharmaceutical agents orcompositions which include polynucleotides encoding any of thesepeptides (i.e., the polynucleotides of this invention).

Cancers to be treated by the pharmaceutical agents or compositions ofthe present invention are not limited and include any cancer in whichTOMM34 is involved (e.g., is over-expressed), including, but not limitedto, AML, CML, bladder cancer, breast cancer, cervical cancer, colorectalcancer, esophagus cancer, liver cancer, osteosarcoma, prostate cancer,renal carcinoma, SCLC, NSCLC and soft tissue tumor.

The pharmaceutical agents or compositions of the present invention maycontain in addition to the aforementioned active ingredients, otherpeptides that have the ability to induce CTLs against cancerous cells,other polynucleotides encoding the other peptides, other cells thatpresent the other peptides, or such. Examples of such “other” peptideshaving the ability to induce CTLs against cancerous cells include, butare not limited to, cancer specific antigens (e.g., identified TAAs).

If necessary, the pharmaceutical agents or compositions of the presentinvention may optionally include other therapeutic substances as anactive ingredient, so long as the substance does not inhibit theantitumoral effect of the active ingredient, e.g., any of the presentpeptides. For example, formulations may include anti-inflammatorysubstances or compositions, pain killers, chemotherapeutics, and thelike. In addition to including other therapeutic substances in themedicament itself, the medicaments of the present invention may also beadministered sequentially or concurrently with the one or more otherpharmacologic compositions. The amounts of medicament and pharmacologiccomposition depend, for example, on what type of pharmacologiccomposition(s) is/are used, the disease being treated, and thescheduling and routes of administration.

Those of skill in the art will readily recognize that, in addition tothe ingredients particularly mentioned herein, the pharmaceutical agentsor compositions of the present invention may further include othersubstances conventional in the art having regard to the type offormulation in question (e.g., fillers, binders, diluents, etc.).

In one embodiment of the present invention, the present pharmaceuticalagents or compositions may be packaged in articles of manufacture, e.g.,as kits containing materials useful for treating the pathologicalconditions of the disease to be treated, e.g., cancer. The article ofmanufacture may include a container of any of the present pharmaceuticalagents or compositions with a label. Suitable containers includebottles, vials, and test tubes. The containers may be formed from avariety of materials, such as glass or plastic. The label on thecontainer should indicate the agent or composition is used for treatingor prevention of one or more conditions of the disease. The label mayalso indicate directions for administration and so on.

In addition to the container described above, a kit including apharmaceutical agent or composition of the present invention mayoptionally further include a second container housing apharmaceutically-acceptable diluent. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

The pharmaceutical compositions can, if desired, be presented in a packor dispenser device which can contain one or more unit dosage formscontaining the active ingredient. The pack can, for example, includemetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration.

(1) Pharmaceutical Agents or Compositions Containing the Peptides as theActive Ingredient

The peptides of this invention can be administered directly as apharmaceutical agent or composition, or if necessary, may be formulatedby conventional formulation methods. In the latter case, in addition tothe peptides of this invention, carriers, excipients, and such that areordinarily used for drugs can be included as appropriate withoutparticular limitations. Examples of such carriers are sterilized water,physiological saline, phosphate buffer, culture fluid and such.Furthermore, the pharmaceutical agents or compositions can contain asnecessary, stabilizers, suspensions, preservatives, surfactants andsuch. The pharmaceutical agents or compositions of this invention can beused for anticancer purposes.

The peptides of this invention can be prepared in a combination, whichincludes two or more of peptides of the present invention, to induce CTLin vivo. The peptides can be in a cocktail or can be conjugated to eachother using standard techniques. For example, the peptides can bechemically linked or expressed as a single fusion polypeptide sequencethat may have one or several amino acid as a linker (e.g., Lysinelinker: K. S. Kawamura et al. J. Immunol. 2002, 168: 5709-5715). Thepeptides in the combination can be the same or different. Byadministering the peptides of this invention, the peptides are presentedat a high density by the HLA antigens on APCs, then CTLs thatspecifically react toward the complex formed between the displayedpeptide and the HLA antigen are induced. Alternatively, APCs (e.g., DCs)are removed from subjects and then stimulated by the peptides of thepresent invention to obtain APCs that present any of the peptides ofthis invention on their cell surface. These APCs are readministered tothe subjects to induce CTLs in the subjects, and as a result,aggressiveness towards the tumor-associated endothelium can beincreased.

The pharmaceutical agents or compositions for treating and/or preventionof cancer, that include any peptide of this invention as the activeingredient, can additionally include an adjuvant so that cellularimmunity will be established effectively, or they can be administeredwith other active ingredients, and they can be administered byformulation into granules. An adjuvant refers to a compound thatenhances the immune response against the protein when administeredtogether (or successively) with the protein having immunologicalactivity. An adjuvant that can be applied includes those described inthe literature (Clin Microbiol Rev 1994, 7: 277-89). Exemplary adjuvantsinclude aluminum phosphate, aluminum hydroxide, alum, cholera toxin,salmonella toxin, Incomplete Freund's adjuvant (IFA), Complete Freund'sadjuvant (CFA), IS-COMatrix, GM-CSF, CpG, O/W emulsion, and such, butare not limited thereto.

Furthermore, liposome formulations, granular formulations in which thepeptide is bound to few-micrometers diameter beads, and formulations inwhich a lipid is bound to the peptide may be conveniently used.

In another embodiment of the present invention, the peptides of thepresent invention may also be administered in the form of apharmaceutically acceptable salt. Preferable examples of the saltsinclude salts with an alkali metal, salts with a metal, salts with anorganic base, salts with an organic acid and salts with an inorganicacid. As used herein, the phrase “pharmaceutically acceptable salt”refers to those salts that retain the biological effectiveness andproperties of the compound and that are obtained by reaction withinorganic acids or bases such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike.

In some embodiments, the pharmaceutical agents or compositions of thepresent invention may further include a component which primes CTL.Lipids have been identified as substances or compositions capable ofpriming CTL in vivo against viral antigens. For example, palmitic acidresidues can be attached to the epsilon- and alpha-amino groups of alysine residue and then linked to a peptide of the present invention.The lipidated peptide can then be administered either directly in amicelle or particle, incorporated into a liposome, or emulsified in anadjuvant. As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine(P3CSS) can be used to prime CTL when covalently attached to anappropriate peptide (see, e.g., Deres et al., Nature 1989, 342: 561-4).

Examples of suitable methods of administration include, but are notnecessarily limited to, oral, intradermal, subcutaneous, intramuscular,intraosseous, peritoneal, and intravenous injection, or such, andsystemic administration or local administration to the vicinity of thetargeted sites. The administration can be performed by singleadministration or boosted by multiple administrations. The dose of thepeptides of this invention can be adjusted appropriately according tothe disease to be treated, age of the patient, weight, method ofadministration, and such, and is ordinarily 0.001 mg to 1,000 mg, forexample, 0.01 mg to 100 mg, for example, 0.1 mg to 10 mg, for example,0.5 mg to 5 mg and can be administered once in a few days to few months.One skilled in the art can appropriately select a suitable dose.

(2) Pharmaceutical Agents or Compositions Containing Polynucleotides asthe Active Ingredient

The pharmaceutical agents or compositions of the present invention canalso include nucleic acids encoding the peptides disclosed herein in anexpressible form. Herein, the phrase “in an expressible form” means thatthe polynucleotide, when introduced into a cell, will be expressed invivo as a polypeptide that induces anti-tumor immunity. In anexemplified embodiment, the nucleic acid sequence of the polynucleotideof interest includes regulatory elements necessary for expression of thepolynucleotide. The polynucleotide(s) can be equipped so to achievestable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi M R, Cell 1987, 51: 503-12 for a description of homologousrecombination cassette vectors). See, e.g., Wolff et al., Science 1990,247: 1465-8; U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118;5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based deliverytechnologies include “naked DNA”, facilitated (bupivacaine, polymers,peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g.,U.S. Pat. No. 5,922,687).

The peptides of the present invention can also be expressed by viral orbacterial vectors. Examples of expression vectors include attenuatedviral hosts, such as vaccinia or fowlpox. This approach involves the useof vaccinia virus, e.g., as a vector to express nucleotide sequencesthat encode the peptide. Upon introduction into a host, the recombinantvaccinia virus expresses the immunogenic peptide, and thereby elicits animmune response. Vaccinia vectors and methods useful in immunizationprotocols are described in, e.g., U.S. Pat. No. 4,722,848. Anothervector is BCG (Bacille Calmette Guerin). BCG vectors are described inStover et al., Nature 1991, 351: 456-60. A wide variety of other vectorsuseful for therapeutic administration or immunization e.g., adeno andadeno-associated virus vectors, retroviral vectors, Salmonella typhivectors, detoxified anthrax toxin vectors, and the like, will beapparent. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71;Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo2000, 14: 571-85.

Delivery of a polynucleotide into a patient can be either direct, inwhich case the patient is directly exposed to a polynucleotide-carryingvector, or indirect, in which case, cells are first transformed with thepolynucleotide of interest in vitro, then the cells are transplantedinto the patient. Theses two approaches are known, respectively, as invivo and ex vivo gene therapies.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 1993, 12: 488-505; Wu and Wu, Biotherapy 1991, 3:87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96; Mulligan,Science 1993, 260: 926-32; Morgan & Anderson, Ann Rev Biochem 1993, 62:191-217; Trends in Biotechnology 1993, 11(5): 155-215). Methods commonlyknown in the art of recombinant DNA technology that are applicable tothe present invention are described by Ausubel et al. in CurrentProtocols in Molecular Biology, John Wiley & Sons, N Y, 1993; andKrieger in Gene Transfer and Expression, A Laboratory Manual, StocktonPress, N Y, 1990.

Like administration of peptides, administration of polynucleotides maybe performed oral, intradermal, subcutaneous, intraosseous, peritonealand/or intravenous injection, or such, e.g., systemic administration orlocal administration to the vicinity of the targeted sites finds use.The administration can be performed by single administration or boostedby multiple administrations. The dose of the polynucleotide in thesuitable carrier or cells transformed with the polynucleotide encodingthe peptides of this invention can be adjusted appropriately accordingto the disease to be treated, age of the patient, weight, method ofadministration, and such, and is ordinarily 0.001 mg to 1000 mg, forexample, 0.01 mg to 100 mg, for example, 0.1 mg to 10 mg, for example,0.5 mg to 5 mg and can be administered once every a few days to onceevery few months. One skilled in the art can appropriately select thesuitable dose.

X. METHODS USING THE PEPTIDES, EXOSOMES, APCS AND CTLS

The peptides and polynucleotides of the present invention can be usedfor preparing or inducing APCs and CTLs. The exosomes and APCs of thepresent invention can be also used for inducing CTLs. The peptides,polynucleotides, exosomes and APCs can be used in combination with anyother compounds so long as the additional compounds do not inhibit theirCTL inducibility. Thus, any of the aforementioned pharmaceutical agentsor compositions of the present invention can be used for inducing CTLs.In addition thereto, those including the peptides and polynucleotidescan be also used for inducing APCs as explained below.

(1) Method of Inducing Antigen-Presenting Cells (APCs)

The present invention provides methods of inducing APCs with high CTLinducibility using the peptides or polynucleotides of this invention.

The methods of the present invention include the step of contacting APCswith the peptides of this invention in vitro, ex vivo or in vivo. Forexample, the method contacting APCs with the peptides ex vivo caninclude steps of:

a: collecting APCs from a subject, and

b: contacting the APCs of step a with the peptide of the presentinvention.

The APCs are not limited to a particular kind of cells. Examples of APCsinclude, but are not limited to DCs, Langerhans cells, macrophages, Bcells, and activated T cells, which are known to present proteinaceousantigens on their cell surface so as to be recognized by lymphocytes.Preferably, DCs can be used since they have the strongest CTLinducibility among APCs. Any peptides of the present invention can beused by themselves or with other peptides of this invention.

On the other hand, when the peptides of the present invention areadministered to a subject, the APCs are contacted with the peptides invivo, consequently, the APCs with high CTL inducibility are induced inthe body of the subject. Thus, the method of the present invention mayinclude administering the peptides of this invention to a subject.Similarly, when the polynucleotides of this invention are administeredto a subject in an expressible form, the peptides of this invention areexpressed and contacted with APCs in vivo, consequently, the APCs withhigh CTL inducibility are induced in the body of the subject. Thus,instead of the aforementioned step, the method of the present inventionmay include administering the polynucleotides of this invention to asubject. “Expressible form” is described above in section “IX.Pharmaceutical agents or compositions, (2) Pharmaceutical agents orcompositions containing polynucleotides as the active ingredient”.

Alternatively, the method of the present invention may includeintroducing the polynucleotide of this invention into an APC to inducean APC with CTL inducibility. For example, the method can include stepsof:

a: collecting APCs from a subject, and

b: introducing a polynucleotide encoding the peptide of this invention.

Step b can be performed as described above in section “VI.Antigen-presenting cells”.

Alternatively, the present invention provides a method for preparing anantigen-presenting cell (APC) which specifically induces CTL activityagainst TOMM34, wherein the method includes one of the following steps:

(a) contacting an APC with a peptide of the present invention in vitro,ex vivo or in vivo; and

(b) introducing a polynucleotide encoding a peptide of the presentinvention into an APC.

Alternatively, the present invention provides methods for inducing anAPC having CTL inducibility, wherein the methods include the stepselected from the group consisting of:

(a) contacting an APC with the peptide of the present invention, and

(b) introducing the polynucleotide encoding the peptide of the presentinvention into an APC.

The methods of the present invention can be carried out in vitro, exvivo or in vivo. Preferably, the methods of the present invention can becarried out in vitro or ex vivo. APCs used for induction of APCs havingCTL inducibility can be preferably APCs expressing HLA-A2 antigen. SuchAPCs can be prepared by the methods well-known in the arts fromperipheral blood mononuclear cells (PBMCs) obtained from a subject whoseHLA antigen is HLA-A2. The APCs induced by the method of the presentinvention can be APCs that present a complex of the peptide of thepresent invention and HLA antigen (HLA-A2 antigen) on its surface. WhenAPCs induced by the method of the present invention are administered toa subject in order to induce immune responses against cancer in thesubject, the subject is preferably the same one from whom APCs arederived. However, the subject may be a different one from the APC donorso long as the subject has the same HLA type with the APC donor.

In another embodiment, the present invention provide agents orcompositions for use in inducing an APC having CTL inducibility, andsuch agents or compositions include one or more peptides orpolynucleotides of the present invention.

In another embodiment, the present invention provides the use of thepeptide of the present invention or the polynucleotide encoding thepeptide in the manufacture of an agent or composition formulated forinducing APCs.

Alternatively, the present invention further provides the peptide of thepresent invention or the polypeptide encoding the peptide for use ininducing an APC having CTL inducibility.

(2) Method of Inducing CTLs

The present invention also provides methods for inducing CTLs using thepeptides, polynucleotides, or exosomes or APCs of this invention.

The present invention also provides methods for inducing CTLs using apolynucleotide/polynucleotides encoding polypeptides (i.e., TCRsubunits) that is capable of forming a T cell receptor (TCR) that iscapable of recognizing a complex of the peptides of the presentinvention and HLA antigens. Preferably, the methods for inducing CTLsinclude at least one step selected from among:

a) contacting a CD8-positive T cell with an antigen-presenting celland/or an exosome that presents on its surface a complex of an HLAantigen and a peptide of the preset invention; and

b) introducing a polynucleotide/polynucleotides encoding polypeptidesthat are capable of forming a TCR that is capable of recognizing acomplex of a peptide of the present invention and an HLA antigen into aCD8-positive T cell.

When the peptides, the polynucleotides, APCs, or exosomes of thisinvention are administered to a subject, CTL is induced in the body ofthe subject, and the strength of the immune response targeting thecancer cells is enhanced. Thus, instead of aforementioned step, themethods of the present invention may include the step of administeringthe peptides, the polynucleotides, the APCs or exosomes of thisinvention to a subject.

Alternatively, CTL can be also induced by using them ex vivo, and afterinducing CTL, the activated CTLs are returned to the subject. Forexample, the method can include steps of:

a: collecting APCs from subject,

b: contacting with the APCs of step a, with the peptide of the presentinvention, and

c: co-culturing the APCs of step b with CD8-positive T cells.

The APCs to be co-cultured with the CD8-positive T cells in above step ccan also be prepared by transferring a gene that includes apolynucleotide of this invention into APCs as described above in section“VI. Antigen-presenting cells”, although the present invention is notlimited thereto and encompasses any APC that effectively presents thepresent on its surface a complex of an HLA antigen and a peptide of thisinvention.

One may optionally utilize the exosomes that presents on its surface acomplex of an HLA antigen and the peptide of this invention instead ofthe aforementioned APCs. Namely, the present invention can includes thestep of co-culturing exosomes presenting on its surface a complex of anHLA antigen and the peptide of this invention. Such exosomes can beprepared by the methods described above in section “V. Exosomes”.

Furthermore, the CTL of the present invention can be induced byintroducing into a CD8-positive T cell a polynucleotide/polynucleotidesencoding the TCR subunits, wherein the TCR formed by such TCR subunitsis capable of binding to a complex of an HLA antigen and the peptide ofthis invention on a cell surface. Such transduction can be performed asdescribed above in section “VIII. T cell receptor (TCR)”.

The methods of the present invention can be carried out in vitro, exvivo or in vivo. Preferably, the methods of the present invention can becarried out in vitro or ex vivo. CD8-positive T cells used for inductionof CTLs can be prepared by well-known methods in the art from PBMCsobtained from a subject. In preferred embodiments, the donor forCD8-positive T cells can be a subject whose HLA antigen is HLA-A2. TheCTLs induced by the methods of the present invention can be CTLs thatcan recognize cells presenting a complex of the peptide of the presentinvention and HLA antigen on its surface. When CTLs induced by themethod of the present invention are administered to a subject in orderto induce immune responses against cancer in the subject, the subject ispreferably the same one from whom CD8-positive T cells are derived.However, the subject may be a different one from the CD8-positive T celldonor so long as the subject has the same HLA type with the CD8-positiveT cell donor.

In addition, the present invention provides a method or process formanufacturing a pharmaceutical composition inducing CTLs, wherein themethod includes the step of admixing or formulating the peptide of thepresent invention with a pharmaceutically acceptable carrier.

In another embodiment, the present invention provide an agent orcomposition for inducing CTL, wherein the agent or composition comprisesone or more peptide(s), one or more polynucleotide(s), or one o moreAPCs or exosomes of the present invention.

In another embodiment, the present invention provides the use of thepeptide, the polynucleotide, or APC or exosome of the present inventionin the manufacture of an agent or composition formulated for inducing aCTL.

Alternatively, the present invention further provides the peptide, thepolynucleotide, or APC or exosome of the present invention for use ininducing a CTL.

(3) Method of Inducing Immune Response

Moreover, the present invention provides methods for an inducing immuneresponse against diseases related to TOMM34. Suitable diseases includecancer, examples of which include, but are not limited to, AML, CML,bladder cancer, breast cancer, cervical cancer, colorectal cancer,esophagus cancer, liver cancer, osteosarcoma, prostate cancer, renalcarcinoma, SCLC, NSCLC and soft tissue tumor.

The methods of the present invention include the step of administeringagents or compositions containing any of the peptides of the presentinvention or polynucleotides encoding them. Alternatively, the method ofthe present invention may include the step of administering exosomes orAPCs presenting any of the peptides of the present invention. Fordetails, see the item of “IX. Pharmaceutical agents or compositions”,particularly the part describing the use of the pharmaceutical agents orcompositions of the present invention as vaccines. In addition, theexosomes and APCs that can be employed for the present methods forinducing immune response are described in detail under the items of “V.Exosomes”, “VI. Antigen-presenting cells (APCs)”, and (1) and (2) of “X.Methods using the peptides, exosomes, APCs and CTLs”, supra.

The present invention also provides a method or process formanufacturing a pharmaceutical agent or composition inducing immuneresponse, wherein the method includes the step of admixing orformulating the peptide of the present invention with a pharmaceuticallyacceptable carrier.

Alternatively, the method of the present invention may include the stepof administrating a vaccine or a pharmaceutical composition of thepresent invention that contains:

(a) a peptide of the present invention;

(b) a nucleic acid encoding such a peptide as disclosed herein in anexpressible form;

(c) an APC or an exosome presenting a peptide of the present inventionon its surface; or

(d) a cytotoxic T cell of the present invention.

In the context of the present invention, cancer over-expressing TOMM34can be treated with these active ingredients. Examples of such cancerincludes, but is not limited to, AML, CML, bladder cancer, breastcancer, cervical cancer, colorectal cancer, esophagus cancer, livercancer, osteosarcoma, prostate cancer, renal carcinoma, SCLC, NSCLC andsoft tissue tumor. Accordingly, prior to the administration of thevaccines or pharmaceutical compositions including the activeingredients, it is preferable to confirm whether the expression level ofTOMM34 in the cells or tissues to be treated is enhanced compared withnormal cells of the same organ. Thus, in one embodiment, the presentinvention provides a method for treating cancer (over)expressing TOMM34,which method may include the steps of:

i) determining the expression level of TOMM34 in cells or tissue(s)obtained from a subject with the cancer to be treated;

ii) comparing the expression level of TOMM34 with normal control; and

iii) administrating at least one component selected from among (a) to(d) described above to a subject with cancer over-expressing TOMM34compared with normal control.

Alternatively, the present invention provides a vaccine orpharmaceutical composition that includes at least one component selectedfrom among (a) to (d) described above, for use in administrating to asubject having cancer over-expressing TOMM34. In other words, thepresent invention further provides a method for identifying a subject tobe treated with a TOMM34 polypeptide of the present invention, suchmethod including the step of determining an expression level of TOMM34in subject-derived cells or tissue(s), wherein an increase of the levelcompared to a normal control level of the gene indicates that thesubject has cancer which may be treated with the TOMM34 polypeptide ofthe present invention. The methods of treating cancer of the presentinvention are described in more detail below.

Any subject-derived cell or tissue can be used for the determination ofTOMM34 expression so long as it includes the objective transcription ortranslation product of TOMM34. Examples of suitable samples include, butare not limited to, bodily tissues and fluids, such as blood, sputum andurine. Preferably, the subject-derived cell or tissue sample contains acell population including an epithelial cell, more preferably acancerous epithelial cell or an epithelial cell derived from tissuesuspected to be cancerous. Further, if necessary, the cell may bepurified from the obtained bodily tissues and fluids, and then used asthe subjected-derived sample.

A subject to be treated by the present method is preferably a mammal.Exemplary mammals include, but are not limited to, e.g., human,non-human primate, mouse, rat, dog, cat, horse, and cow.

According to the present invention, the expression level of TOMM34 incells or tissues obtained from a subject is determined. The expressionlevel can be determined at the transcription (nucleic acid) productlevel, using methods known in the art. For example, the mRNA of TOMM34may be quantified using probes by hybridization methods (e.g., Northernhybridization). The detection may be carried out on a chip or an array.The use of an array is preferable for detecting the expression level ofTOMM34. Those skilled in the art can prepare such probes utilizing thesequence information of TOMM34. For example, the cDNA of TOMM34 may beused as the probes. If necessary, the probes may be labeled with asuitable label, such as dyes, fluorescent substances and isotopes, andthe expression level of the gene may be detected as the intensity of thehybridized labels.

Furthermore, the transcription product of TOMM34 may be quantified usingprimers by amplification-based detection methods (e.g., RT-PCR). Suchprimers may be prepared based on the available sequence information ofthe gene.

Specifically, a probe or primer used for the present method hybridizesunder stringent, moderately stringent, or low stringent conditions tothe mRNA of TOMM34. As used herein, the phrase “stringent(hybridization) conditions” refers to conditions under which a probe orprimer will hybridize to its target sequence, but not to othersequences. Stringent conditions are sequence-dependent and will bedifferent under different circumstances. Specific hybridization oflonger sequences is observed at higher temperatures than shortersequences. Generally, the temperature of a stringent condition isselected to be about 5 degree Centigrade lower than the thermal meltingpoint (Tm) for a specific sequence at a defined ionic strength and pH.The Tm is the temperature (under a defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary totheir target sequence hybridize to the target sequence at equilibrium.Since the target sequences are generally present at excess, at Tm, 50%of the probes are occupied at equilibrium. Typically, stringentconditions will be those in which the salt concentration is less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30degree Centigrade for short probes or primers (e.g., 10 to 50nucleotides) and at least about 60 degree Centigrade for longer probesor primers. Stringent conditions may also be achieved with the additionof destabilizing substances, such as formamide.

A probe or primer of the present invention is typically a substantiallypurified oligonucleotide. The oligonucleotide typically includes aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100,50, or 25, consecutive sense strand nucleotide sequence of a nucleicacid including a TOMM34 sequence, or an anti sense strand nucleotidesequence of a nucleic acid including a TOMM34 sequence, or of anaturally occurring mutant of these sequences. In particular, forexample, in a preferred embodiment, an oligonucleotide having 5-50 inlength can be used as a primer for amplifying the genes, to be detected.More preferably, mRNA or cDNA of a TOMM34 gene can be detected witholigonucleotide probe or primer of a specific size, generally 15-30b inlength. The size may range from at least 10 nucleotides, at least 12nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least25 nucleotides, at least 30 nucleotides and the probes and primers mayrange in size from 5-10 nucleotides, 10-15 nucleotides, 15-20nucleotides, 20-25 nucleotides and 25-30 nucleotides. In preferredembodiments, length of the oligonucleotide probe or primer can beselected from 15-25. Assay procedures, devices, or reagents for thedetection of gene by using such oligonucleotide probe or primer are wellknown (e.g. oligonucleotide microarray or PCR). In these assays, probesor primers can also include tag or linker sequences. Further, probes orprimers can be modified with detectable label or affinity ligand to becaptured. Alternatively, in hybridization based detection procedures, apolynucleotide having a few hundreds (e.g., about 100-200) bases to afew kilo (e.g., about 1000-2000) bases in length can also be used for aprobe (e.g., northern blotting assay or cDNA microarray analysis).

Alternatively, the translation product may be detected for the diagnosisof the present invention. For example, the quantity of TOMM34 protein(SEQ ID NO: 42) or the immunologically fragment thereof may bedetermined. Methods for determining the quantity of the protein as thetranslation product include immunoassay methods that use an antibodyspecifically recognizing the protein. The antibody may be monoclonal orpolyclonal. Furthermore, any fragment or modification (e.g., chimericantibody, scFv, Fab, F(ab′)₂, Fv, etc.) of the antibody may be used forthe detection, so long as the fragment or modified antibody retains thebinding ability to the TOMM34 protein. Such antibodies against thepeptides of the present invention and the fragments thereof are alsoprovided by the present invention. Methods to prepare these kinds ofantibodies for the detection of proteins are well known in the art, andany method may be employed in the present invention to prepare suchantibodies and equivalents thereof.

As another method to detect the expression level of TOMM34 gene based onits translation product, the intensity of staining may be measured viaimmunohistochemical analysis using an antibody against the TOMM34protein. Namely, in this measurement, strong staining indicatesincreased presence/level of the protein and, at the same time, highexpression level of TOMM34 gene.

The expression level of a target gene, e.g., the TOMM34 gene, in cancercells can be determined to be increased if the level increases from thecontrol level (e.g., the level in normal cells) of the target gene by,for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, morethan 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0fold, or more.

In the context of the present invention, a control level determined froma biological sample that is known to be non-cancerous is referred to asa “normal control level”. On the other hand, if the control level isdetermined from a cancerous biological sample, it is referred to as a“cancerous control level”. Difference between a sample expression leveland a control level can be normalized to the expression level of controlnucleic acids, e.g., housekeeping genes, whose expression levels areknown not to differ depending on the cancerous or non-cancerous state ofthe cell. Exemplary control genes include, but are not limited to,beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomalprotein P1.

The control level may be determined at the same time with the cancercells by using a sample(s) previously collected and stored from asubject/subjects whose disease state(s) (cancerous or non-cancerous)is/are known. In addition, normal cells obtained from non-cancerousregions of an organ that has the cancer to be treated may be used asnormal control. Alternatively, the control level may be determined by astatistical method based on the results obtained by analyzing previouslydetermined expression level(s) of TOMM34 gene in samples from subjectswhose disease states are known. Furthermore, the control level can bederived from a database of expression patterns from previously testedcells. Moreover, according to an aspect of the present invention, theexpression level of TOMM34 gene in a biological sample may be comparedto multiple control levels, determined from multiple reference samples.It is preferred to use a control level determined from a referencesample derived from a tissue type similar to that of the subject-derivedbiological sample. Moreover, it is preferred to use the standard valueof the expression levels of TOMM34 gene in a population with a knowndisease state. The standard value may be obtained by any method known inthe art. For example, a range of mean+/−2 S.D. or mean+/−3 S.D. may beused as the standard value.

In the context of the present invention, a control level determined froma biological sample that is known to be non-cancerous is referred to asa “normal control level”. On the other hand, if the control level isdetermined from a cancerous biological sample, it is referred to as a“cancerous control level”.

When the expression level of TOMM34 gene is increased as compared to thenormal control level, or is similar/equivalent to the cancerous controllevel, the subject may be diagnosed with cancer to be treated.

The present invention also provides a method of (i) diagnosing whether asubject has the cancer to be treated, and/or (ii) selecting a subjectfor cancer treatment, which method includes the steps of:

a) determining the expression level of TOMM34 in cells or tissue(s)obtained from a subject who is suspected to have the cancer to betreated;

b) comparing the expression level of TOMM34 with a normal control level;

c) diagnosing the subject as having the cancer to be treated, if theexpression level of TOMM34 is increased as compared to the normalcontrol level; and

d) selecting the subject for cancer treatment, if the subject isdiagnosed as having the cancer to be treated, in step c).

Alternatively, such a method includes the steps of:

a) determining the expression level of TOMM34 in cells or tissue(s)obtained from a subject who is suspected to have the cancer to betreated;

b) comparing the expression level of TOMM34 with a cancerous controllevel;

c) diagnosing the subject as having the cancer to be treated, if theexpression level of TOMM34 is similar or equivalent to the cancerouscontrol level; and

d) selecting the subject for cancer treatment, if the subject isdiagnosed as having the cancer to be treated, in step c).

The present invention also provides a diagnostic kit for diagnosing ordetermining a subject who is or is suspected to be suffering from cancerthat can be treated with the TOMM34 polypeptide of the presentinvention, which may also be useful in assessing the prognosis of cancerand/or monitoring the efficacy or applicability of a cancer therapy,particularly a cancer immunotherapy. Illustrative examples of suitablecancers include, but are not limited to, AML, CML, bladder cancer,breast cancer, cervical cancer, colorectal cancer, esophagus cancer,liver cancer, osteosarcoma, prostate cancer, renal carcinoma, SCLC,NSCLC and soft tissue tumor. More particularly, the kit preferablyincludes at least one reagent for detecting the expression of the TOMM34gene in a subject-derived cell, such reagent selected from the group of:

(a) a reagent for detecting mRNA of the TOMM34 gene;

(b) a reagent for detecting the TOMM34 protein or the immunologicallyfragment thereof; and

(c) a reagent for detecting the biological activity of the TOMM34protein.

Examples of reagents suitable for detecting mRNA of the TOMM34 geneinclude nucleic acids that specifically bind to or identify the TOMM34mRNA, such as oligonucleotides that have a complementary sequence to aportion of the TOMM34 mRNA. These kinds of oligonucleotides areexemplified by primers and probes that are specific to the TOMM34 mRNA.These kinds of oligonucleotides may be prepared based on methods wellknown in the art. If needed, the reagent for detecting the TOMM34 mRNAmay be immobilized on a solid matrix. Moreover, more than one reagentfor detecting the TOMM34 mRNA may be included in the kit.

On the other hand, examples of reagents suitable for detecting theTOMM34 protein or the immunologically fragment thereof may includeantibodies to the TOMM34 protein or the immunologically fragmentthereof. The antibody may be monoclonal or polyclonal. Furthermore, anyfragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab′)₂,Fv, etc.) of the antibody may be used as the reagent, so long as thefragment or modified antibody retains the binding ability to the TOMM34protein or the immunologically fragment thereof. Methods to preparethese kinds of antibodies for the detection of proteins are well knownin the art, and any method may be employed in the present invention toprepare such antibodies and equivalents thereof. Furthermore, theantibody may be labeled with signal generating molecules via directlinkage or an indirect labeling technique. Labels and methods forlabeling antibodies and detecting the binding of the antibodies to theirtargets are well known in the art, and any labels and methods may beemployed for the present invention. Moreover, more than one reagent fordetecting the TOMM34 protein may be included in the kit.

The kit may contain more than one of the aforementioned reagents. Thekit can further include a solid matrix and reagent for binding a probeagainst a TOMM34 gene or antibody against a TOMM34 peptide, a medium andcontainer for culturing cells, positive and negative control reagents,and a secondary antibody for detecting an antibody against a TOMM34peptide. For example, tissue samples obtained from subjects withoutcancer or suffering from cancer, may serve as useful control reagents. Akit of the present invention may further include other materialsdesirable from a commercial and user standpoint, including buffers,diluents, filters, needles, syringes, and package inserts (e.g.,written, tape, CD-ROM, etc.) with instructions for use. These reagentsand such may be retained in a container with a label. Suitablecontainers include bottles, vials, and test tubes. The containers may beformed from a variety of materials, such as glass or plastic.

In one embodiment of the present invention, when the reagent is a probeagainst the TOMM34 mRNA, the reagent may be immobilized on a solidmatrix, such as a porous strip, to form at least one detection site. Themeasurement or detection region of the porous strip may include aplurality of sites, each containing a nucleic acid (probe). A test stripmay also contain sites for negative and/or positive controls.Alternatively, control sites may be located on a strip separated fromthe test strip. Optionally, the different detection sites may containdifferent amounts of immobilized nucleic acids, i.e., a higher amount inthe first detection site and lesser amounts in subsequent sites. Uponthe addition of a test sample, the number of sites displaying adetectable signal provides a quantitative indication of the amount ofTOMM34 mRNA present in the sample. The detection sites may be configuredin any suitably detectable shape and are typically in the shape of a baror dot spanning the width of a test strip.

The kit of the present invention may further include a positive controlsample or TOMM34 standard sample. The positive control sample of thepresent invention may be prepared by collecting TOMM34 positive samplesand then assaying their TOMM34 levels. Alternatively, a purified TOMM34protein or polynucleotide may be added to cells that do not expressTOMM34 to form the positive sample or the TOMM34 standard sample. In thepresent invention, purified TOMM34 may be a recombinant protein. TheTOMM34 level of the positive control sample is, for example, more thanthe cut off value.

In one embodiment, the present invention further provides a diagnostickit including, a protein or a partial protein thereof specificallyrecognized by the antibody of the present invention or the fragmentthereof.

Examples of the partial peptide of the protein of the present inventioninclude polypeptides consisting of at least 8, preferably 15, and morepreferably 20 contiguous amino acids in the amino acid sequence of theprotein of the present invention. Cancer can be diagnosed by detectingan antibody in a sample (e.g., blood, tissue) using a protein or apeptide (polypeptide) of the present invention. The method for preparingthe protein of the present invention and peptides are as describedabove.

The present invention provides methods for diagnosing cancer, which canbe performed by determining the difference between the amount ofanti-TOMM34 antibody and that in the corresponding control sample asdescribe above. The subject is suspected to be suffering from cancer, ifcells or tissues of the subject contain antibodies against theexpression products (TOMM34) of the gene and the quantity of theanti-TOMM34 antibody is determined to be more than the cut off value inlevel compared to that in normal control.

In another embodiment, a diagnostic kit of the present invention mayinclude the peptide of the present invention and an HLA molecule bindingthereto. The method for detecting antigen specific CTLs using antigenicpeptides and HLA molecules has already been established (for example,Altman J D et al., Science. 1996, 274(5284): 94-6). Thus, the complex ofthe peptide of the present invention and the HLA molecule can be appliedto the detection method to detect tumor antigen specific CTLs, therebyenabling earlier detection, recurrence and/or metastasis of cancer.Further, it can be employed for the selection of subjects applicablewith the pharmaceuticals including the peptide of the present inventionas an active ingredient, or the assessment of the treatment effect ofthe pharmaceuticals.

Particularly, according to the known method (see, for example, Altman JD et al., Science. 1996, 274(5284): 94-6), the oligomer complex, such astetramer, of the radiolabeled HLA molecule and the peptide of thepresent invention can be prepared. With using the complex, the diagnosiscan be done, for example, by quantifying the antigen-peptide specificCTLs in the peripheral blood lymphocytes derived from the subjectsuspected to be suffering from cancer.

The present invention further provides a method or diagnostic agents forevaluating immunological response of subject by using peptide epitopesas described herein. In one embodiment of the invention, HLA A-2restricted peptides as described herein are used as reagents forevaluating or predicting an immune response of a subject. The immuneresponse to be evaluated is induced by contacting an immunogen withimmunocompetent cells in vitro or in vivo. In preferred embodiments, theimmunocompetent cells for evaluating an immunological response, may beselected among peripheral blood, peripheral blood lymphocyte (PBL), andperipheral blood mononuclear cell (PBMC). Methods for collecting orisolating such immunocompetent cells are well known in the arts. In someembodiments, any agent that may result in the production of antigenspecific CTLs that recognize and bind to the peptide epitope (s) may beemployed as the reagent. The peptide reagent need not be used as theimmunogen. Assay systems that are used for such an analysis includerelatively recent technical developments such as tetramers, staining forintracellular lymphokines and interferon release assays, or ELISPOTassays. In a preferred embodiment, immunocompetent cells to be contactedwith peptide reagent may be antigen presenting cells including dendriticcells.

For example, peptides of the present invention may be used in tetramerstaining assays to assess peripheral blood mononuclear cells for thepresence of antigen-specific CTLs following exposure to a tumor cellantigen or an immunogen. The HLA tetrameric complex may be used todirectly visualize antigen specific CTLs (see, e.g., Ogg et al., Science279: 2103-2106, 1998; and Altman et al, Science 174: 94-96, 1996) anddetermine the frequency of the antigen-specific CTL population in asample of peripheral blood mononuclear cells. A tetramer reagent using apeptide of the invention may be generated as follows:

A peptide that binds to an HLA molecule is refolded in the presence ofthe corresponding HLA heavy chain and beta 2-microglobulin to generate atrimolecular complex. In the complex, carboxyl terminal of the heavychain is biotinylated at a site that was previously engineered into theprotein. Then, streptavidin is added to the complex to form tetramercomposed of the trimolecular complex and streptavidin. By means offluorescently labeled streptavidin, the tetramer can be used to stainantigen-specific cells. The cells can then be identified, for example,by flow cytometry. Such an analysis may be used for diagnostic orprognostic purposes. Cells identified by the procedure can also be usedfor therapeutic purposes.

The present invention also provides reagents to evaluate immune recallresponses (see, e.g., Bertoni et aL, J. Clin. Invest. 100: 503-513, 1997and Penna et aL, J Exp. Med. 174: 1565-1570, 1991) comprising peptidesof the present invention. For example, patient PBMC samples fromindividuals with cancer to be treated are analyzed for the presence ofantigen-specific CTLs using specific peptides. A blood sample containingmononuclear cells can be evaluated by cultivating the PBMCs andstimulating the cells with a peptide of the invention. After anappropriate cultivation period, the expanded cell population can beanalyzed, for example, for CTL activity.

The peptides may be also used as reagents to evaluate the efficacy of avaccine. PBMCs obtained from a patient vaccinated with an immunogen maybe analyzed using, for example, either of the methods described above.The patient is HLA typed, and peptide epitope reagents that recognizethe allele specific molecules present in that patient are selected forthe analysis. The immunogenicity of the vaccine may be indicated by thepresence of epitope-specific CTLs in the PBMC sample.

The peptides of the invention may be also used to make antibodies, usingtechniques well known in the art (see, e.g.CURRENTPROTOCOLSINIMMUNOLOGY, Wiley/Greene, NY; and Antibodies ALaboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press,1989), which may be useful as reagents to diagnose or monitor cancer.Such antibodies may include those that recognize a peptide in thecontext of an HLA molecule, i.e., antibodies that bind to a peptide-MHCcomplex.

The peptides and compositions of the present invention have a number ofadditional uses, some of which are described herein. For instance, thepresent invention provides a method for diagnosing or detecting adisorder characterized by expression of a TOMM34 immunogenicpolypeptide. These methods include the step of determining expressionlevel of the peptide of the present invention, or a complex of thepeptide of the present invention and an HLA class I molecule in abiological sample. The expression of a peptide or complex of peptide andHLA class I molecule can be determined or detected by assaying with abinding partner for the peptide or complex. In an preferred embodiment,a binding partner for the peptide or complex is an antibody recognizesand specifically bind to the peptide. The expression of TOMM34 in abiological sample, such as a tumor biopsy, can also be tested bystandard PCR amplification protocols using TOMM34 primers. An example oftumor expression is presented herein and further disclosure of exemplaryconditions and primers for TOMM34 amplification can be found inWO2003/27322.

Preferably, the diagnostic methods include the step of contacting abiological sample isolated from a subject with an agent specific for thepeptide of the present invention to detect the presence of the peptideof the present invention in the biological sample. As used herein,“contacting” means placing the biological sample in sufficient proximityto the agent and under the appropriate conditions of, e.g.,concentration, temperature, time, ionic strength, to allow the specificinteraction between the agent and the peptide of the present inventionthat are present in the biological sample. In general, the conditionsfor contacting the agent with the biological sample are conditions knownby those of ordinary skill in the art to facilitate a specificinteraction between a molecule and its cognate (e.g., a protein and itsreceptor, an antibody and its protein antigen, a nucleic acid and itscomplementary strand) in a biological sample. Optimal conditions forfacilitating a specific interaction between a molecule and its cognateare described in U.S. Pat. No. 5,108,921, issued to Low et al.

The diagnostic method of the present invention can be performed ineither or both of in vivo and in vitro. Accordingly, biological samplecan be located in vivo or in vitro in the present invention. Forexample, the biological sample can be a tissue in vivo and the agentspecific for the TOMM34 immunogenic polypeptide can be used to detectthe presence of such molecules in the tissue. Alternatively, thebiological sample can be collected or isolated in vitro (e.g., a bloodsample, tumor biopsy specimen, tissue extract). In a particularlypreferred embodiment, the biological sample can be a cell-containingsample, more preferably a sample containing tumor cells collected from asubject to be diagnosed or treated.

Alternatively, the diagnosis can be done, by a method which allowsdirect quantification of antigen-specific T cells by staining withFluorescein-labelled HLA multimeric complexes (for example, Altman, J.D. et al., 1996, Science 274: 94; Altman, J. D. et al., 1993, Proc.Natl. Acad. Sci. USA 90: 10330). Staining for intracellular lymphokines,and interferon-gamma release assays or ELISPOT assays also has beenprovided. Tetramer staining, intracellular lymphokine staining andELISPOT assays all appear to be at least 10-fold more sensitive thanmore conventional assays (Murali-Krishna, K. et al., 1998, Immunity 8:177; Lalvani, A. et al., 1997, J. Exp. Med. 186: 859; Dunbar, P. R. etal., 1998, Curr. Biol. 8: 413). Pentamers (e.g., US 2004-209295A),dextramers (e.g., WO 02/072631), and streptamers (e.g., Nature medicine6. 631-637 (2002)) may also be used.

For instance, in some embodiments, the present invention provides amethod for diagnosing or evaluating an immunological response of asubject administered at least one of TOMM34 peptides of the presentinvention, the method including the steps of:

(a) contacting an immunogen with immunocompetent cells under thecondition suitable

of induction of CTL specific to the immunogen;

(b) detecting or determining induction level of the CTL induced in step(a); and

(c) correlating the immunological response of the subject with the CTLinduction level.

In the present invention, the immunogen preferably includes at least oneof (a) TOMM34 peptide(s) of the present invention, for example, peptideshaving the amino acid sequence selected from among SEQ ID NOs: 1, 5, 31and 32, and modified peptides thereof (peptides having such amino acidsequences, and peptides having in which such amino acid sequences havebeen modified with 1, 2 or more amino acid substitution(s)). In themeantime, conditions suitable of induction of immunogen specific CTL arewell known in the art. For example, immunocompetent cells may becultured in vitro under the presence of immunogen(s) to induce immunogenspecific CTL. In order to induce immunogen specific CTLs, anystimulating factors may be added to the cell culture. For example, IL-2is preferable stimulating factors for the CTL induction. In someembodiments, the step of monitoring or evaluating immunological responseof a subject to be treated with peptide cancer therapy may be performedbefore, during and/or after the treatment. In general, during a protocolof cancer therapy, immunogenic peptides are administered repeatedly to asubject to be treated. For example, immunogenic peptides may beadministered every week for 3-10 weeks. Accordingly, the immunologicalresponse of the subject can be evaluated or monitored during the cancertherapy protocol. Alternatively, the step of evaluation or monitoring ofimmunological response to the cancer therapy may at the completion ofthe therapy protocol. According to the present invention, enhancedinduction of immunogen specific CTL as compared with a control indicatesthat the subject to be evaluated or diagnosed immunologically respondedto the immunogen(s) that has/have been administered. Suitable controlsfor evaluating the immunological response may include, for example, aCTL induction level when the immunocompetent cells are contacted with nopeptide, or control peptide(s) having amino acid sequences other thanany TOMM34 peptides. (e.g. random amino acid sequence). In a preferredembodiment, the immunological response of the subject is evaluated in asequence specific manner, by comparison with an immunological responsebetween each immunogen administered to the subject. In particular, evenwhen a mixture of some kinds of TOMM34 peptides is administered to thesubject, immunological response might vary depending on the peptides. Inthat case, by comparison of the immunological response between eachpeptide, peptides to which the subject show higher response can beidentified.

XI. ANTIBODIES

The present invention provides antibodies that bind to the peptide ofthe present invention. Preferred antibodies specifically bind to thepeptide of the present invention and will not bind (or will bind weakly)to non-peptide of the present invention. Alternatively, antibodies bindthe peptide of the invention as well as the homologs thereof.

Antibodies against the peptide of the invention can find use in cancerdiagnostic and prognostic assays, and imaging methodologies. Similarly,such antibodies can find use in the treatment, diagnosis, and/orprognosis of other cancers, to the extent TOMM34 is also expressed orover-expressed in cancer patient. Moreover, intracellularly expressedantibodies (e.g., single chain antibodies) are therapeutically useful intreating cancers in which the expression of TOMM34 is involved, examplesof which include, but are not limited to AML, CML, bladder cancer,breast cancer, cervical cancer, colorectal cancer, esophagus cancer,liver cancer, osteosarcoma, prostate cancer, renal carcinoma, SCLC,NSCLC and soft tissue tumor.

The present invention also provides various immunological assay for thedetection and/or quantification of the TOMM34 protein (SEQ ID NO: 42) orfragments thereof including the peptides of the present invention. Suchassays can comprise one or more anti-TOMM34 antibodies capable ofrecognizing and binding a TOMM34 protein or fragments thereof, asappropriate. In the context of the present invention, anti-TOMM34antibodies binding to TOMM34 polypeptide preferably recognize a peptideof the present invention. A binding specificity of antibody can beconfirmed with inhibition test. That is, when the binding between anantibody to be analyzed and full-length of TOMM34 polypeptide wasinhibited under presence of a peptide of the present invention, it isshown that this antibody specifically binds to the fragment. In thecontext of the present invention, such immunological assays areperformed within various immunological assay formats well known in theart, including but not limited to various types of radioimmunoassays,immuno-chromatograph technique, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Related immunological but non-antibody assays of the invention alsocomprise T cell immunogenicity assays (inhibitory or stimulatory) aswell as major histocompatibility complex (MHC) binding assays. Inaddition, immunological imaging methods capable of detecting cancersexpressing TOMM34 are also provided by the invention, including but notlimited to radioscintigraphic imaging methods using labeled antibodiesof the present invention. Such assays are clinically useful in thedetection, monitoring, and prognosis of TOMM34 expressing cancers,examples of which include, but are not limited to, AML, CML, bladdercancer, breast cancer, cervical cancer, colorectal cancer, esophaguscancer, liver cancer, osteosarcoma, prostate cancer, renal carcinoma,SCLC, NSCLC and soft tissue tumor.

The present invention provides antibodies that bind to the peptide ofthe invention. An antibody of the invention can be used in any form,such as monoclonal or polyclonal antibodies, and includes antiserumobtained by immunizing an animal such as a rabbit with the peptide ofthe invention, all classes of polyclonal and monoclonal antibodies,human antibodies and humanized antibodies produced by geneticrecombination.

A peptide of the invention used as an antigen to obtain an antibody maybe derived from any animal species, but preferably is derived from amammal such as a human, mouse, or rat, more preferably from a human. Ahuman-derived peptide may be obtained from the nucleotide or amino acidsequences disclosed herein.

According to the present invention, the peptide to be used as animmunization antigen may be a complete protein or a partial peptide ofthe protein. A partial peptide may comprise, for example, the amino(N)-terminal or carboxy (C)-terminal fragment of a peptide of thepresent invention.

Herein, an antibody of the present invention is defined as a proteinthat reacts with either the full length or a fragment of a TOMM34peptide. In a preferred embodiment, an antibody of the present inventionrecognizes fragment peptides of TOMM34 having an amino acid sequenceselected from among SEQ ID NOs: 1, 5, 31 and 32. Methods forsynthesizing oligopeptide are well known in the arts. After thesynthesis, peptides may be optionally purified prior to use asimmunogen. In the present invention, the oligopeptide (e.g. 9 or 10 mer)may be conjugated or linked with carriers to enhance the immunogenicity.Keyhole-limpet hemocyanin (KLH) is well known as the carrier. Method forconjugating KLH and peptide are also well known in the arts.

Alternatively, a gene encoding a peptide of the invention or itsfragment may be inserted into a known expression vector, which is thenused to transform a host cell as described herein. The desired peptideor its fragment may be recovered from the outside or inside of hostcells by any standard method, and may subsequently be used as anantigen. Alternatively, whole cells expressing the peptide or theirlysates or a chemically synthesized peptide may be used as the antigen.

Any mammalian animal may be immunized with the antigen, but preferablythe compatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorpha or Primates areused. Animals of Rodentia include, for example, mouse, rat and hamster.Animals of Lagomorpha include, for example, rabbit. Animals of Primatesinclude, for example, a monkey of Catarrhini (old world monkey) such asMacaca fascicularis, rhesus monkey, sacred baboon and chimpanzees.

Methods for immunizing animals with antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is astandard method for the immunization of mammals. More specifically,antigens may be diluted and suspended in an appropriate amount ofphosphate buffered saline (PBS), physiological saline, etc. If desired,the antigen suspension may be mixed with an appropriate amount of astandard adjuvant, such as Freund's complete adjuvant, made intoemulsion and then administered to mammalian animals. Preferably, it isfollowed by several administrations of antigen mixed with anappropriately amount of Freund's incomplete adjuvant every 4 to 21 days.An appropriate carrier may also be used for immunization. Afterimmunization as above, serum is examined by a standard method for anincrease in the amount of desired antibodies.

Polyclonal antibodies against the peptides of the present invention maybe prepared by collecting blood from the immunized mammal examined forthe increase of desired antibodies in the serum, and by separating serumfrom the blood by any conventional method. Polyclonal antibodies mayinclude serum containing the polyclonal antibodies, as well as thefraction containing the polyclonal antibodies may be isolated from theserum. Immunoglobulin G or M can be prepared from a fraction whichrecognizes only the peptide of the present invention using, for example,an affinity column coupled with the peptide of the present invention,and further purifying this fraction using protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from themammal immunized with the antigen and checked for the increased level ofdesired antibodies in the serum as described above, and are subjected tocell fusion. The immune cells used for cell fusion are preferablyobtained from spleen. Other preferred parental cells to be fused withthe above immunocyte include, for example, myeloma cells of mammalians,and more preferably myeloma cells having an acquired property for theselection of fused cells by drugs.

The above immunocyte and myeloma cells can be fused according to knownmethods, for example, the method of Milstein et al. (Galfre andMilstein, Methods Enzymol 73: 3-46 (1981)).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as HAT medium(hypoxanthine, aminopterin and thymidine containing medium). The cellculture is typically continued in the HAT medium for several days toseveral weeks, the time being sufficient to allow all the other cells,with the exception of the desired hybridoma (non-fused cells), to die.Then, the standard limiting dilution is performed to screen and clone ahybridoma cell producing the desired antibody.

In addition to the above method, in which a non-human animal isimmunized with an antigen for preparing hybridoma, human lymphocytessuch as those infected by EB virus may be immunized with a peptide,peptide expressing cells or their lysates in vitro. Then, the immunizedlymphocytes are fused with human-derived myeloma cells that are capableof indefinitely dividing, such as U266, to yield a hybridoma producing adesired human antibody that is able to bind to the peptide can beobtained (Unexamined Published Japanese Patent Application No. (JP-A)Sho 63-17688).

The obtained hybridomas are subsequently transplanted into the abdominalcavity of a mouse and the ascites are extracted. The obtained monoclonalantibodies can be purified by, for example, ammonium sulfateprecipitation, a protein A or protein G column, DEAE ion exchangechromatography or an affinity column to which the peptide of the presentinvention is coupled. The antibody of the present invention can be usednot only for purification and detection of the peptide of the presentinvention, but also as a candidate for agonists and antagonists of thepeptide of the present invention.

Alternatively, an immune cell, such as an immunized lymphocyte,producing antibodies may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

Monoclonal antibodies thus obtained can be also recombinantly preparedusing genetic engineering techniques (see, for example, Borrebaeck andLarrick, Therapeutic Monoclonal Antibodies, published in the UnitedKingdom by MacMillan Publishers LTD (1990)). For example, a DNA encodingan antibody may be cloned from an immune cell, such as a hybridoma or animmunized lymphocyte producing the antibody, inserted into anappropriate vector, and introduced into host cells to prepare arecombinant antibody. The present invention also provides recombinantantibodies prepared as described above.

Furthermore, an antibody of the present invention may be a fragment ofan antibody or modified antibody, so long as it binds to one or more ofthe peptides of the invention. For instance, the antibody fragment maybe Fab, F(ab′)2, Fv or single chain Fv (scFv), in which Fv fragmentsfrom H and L chains are ligated by an appropriate linker (Huston et al.,Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically, anantibody fragment may be generated by treating an antibody with anenzyme, such as papain or pepsin. Alternatively, a gene encoding theantibody fragment may be constructed, inserted into an expression vectorand expressed in an appropriate host cell (see, for example, Co et al.,J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178:476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515(1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al.,Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9:132-7 (1991)).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention provides forsuch modified antibodies. The modified antibody can be obtained bychemically modifying an antibody. These modification methods areconventional in the field.

Alternatively, an antibody of the present invention may be obtained as achimeric antibody, between a variable region derived from nonhumanantibody and the constant region derived from human antibody, or as ahumanized antibody, comprising the complementarity determining region(CDR) derived from nonhuman antibody, the frame work region (FR) and theconstant region derived from human antibody. Such antibodies can beprepared according to known technology. Humanization can be performed bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody (see e.g., Verhoeyen et al., Science239:1534-1536 (1988)). Accordingly, such humanized antibodies arechimeric antibodies, wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

Fully human antibodies comprising human variable regions in addition tohuman framework and constant regions can also be used. Such antibodiescan be produced using various techniques known in the art. For examplein vitro methods involve use of recombinant libraries of human antibodyfragments displayed on bacteriophage (e.g., Hoogenboom & Winter, J. Mol.Biol. 227:381 (1991), Similarly, human antibodies can be made byintroducing a human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. This approach is described, e.g., in U.S. Pat.Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016.

Antibodies obtained as above may be purified to homogeneity. Forexample, the separation and purification of the antibody can beperformed according to the separation and purification methods used forgeneral proteins. For example, the antibody may be separated andisolated by the appropriately selected and combined use of columnchromatographies, such as affinity chromatography, filter,ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis and isoelectric focusing (Antibodies: A LaboratoryManual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)),but are not limited thereto. A protein A column and protein G column canbe used as the affinity column. Exemplary protein A columns to be usedinclude, for example, Hyper D, POROS and Sepharose F.F. (Pharmacia).

Examples of suitable chromatography methods, with the exception ofaffinity includes, for example, ion-exchange chromatography, hydrophobicchromatography, gel filtration, reverse-phase chromatography, adsorptionchromatography and the like (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual. Ed Daniel R. Marshak etal., Cold Spring Harbor Laboratory Press (1996)). The chromatographicprocedures can be carried out by liquid-phase chromatography, such asHPLC and FPLC.

For example, measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and/orimmunofluorescence may be used to measure the antigen binding activityof the antibody of the invention. In ELISA, the antibody of the presentinvention is immobilized on a plate, a peptide of the invention isapplied to the plate, and then a sample containing a desired antibody,such as culture supernatant of antibody producing cells or purifiedantibodies, is applied. Then, a secondary antibody that recognizes theprimary antibody and is labeled with an enzyme, such as alkalinephosphatase, is applied, and the plate is incubated. Next, afterwashing, an enzyme substrate, such as p-nitrophenyl phosphate, is addedto the plate, and the absorbance is measured to evaluate the antigenbinding activity of the sample. A fragment of the peptide, such as aC-terminal or N-terminal fragment, may be used as the antigen toevaluate the binding activity of the antibody. BIAcore (Pharmacia) maybe used to evaluate the activity of the antibody according to thepresent invention.

The above methods allow for the detection or measurement of a peptide ofthe invention, by exposing an antibody of the invention to a samplepresumed to contain a peptide of the invention, and detecting ormeasuring the immune complex formed by the antibody and the peptide.

Because the method of detection or measurement of the peptide accordingto the invention can specifically detect or measure a peptide, themethod may be useful in a variety of experiments in which the peptide isused.

XII. VECTORS AND HOST CELLS

The present invention also provides a vector and host cell into which anucleotide encoding the peptide of the present invention is introduced.A vector of the present invention is useful to keep a nucleotide,especially a DNA, of the present invention in host cell, to express thepeptide of the present invention, or to administer the nucleotide of thepresent invention for gene therapy.

When E. coli is a host cell and the vector is amplified and produced ina large amount in E. coli (e.g., JM109, DH5 alpha, HB101 or XL1Blue),the vector should have “ori” to be amplified in E. coli and a markergene for selecting transformed E. coli (e.g., a drug-resistance geneselected by a drug such as ampicillin, tetracycline, kanamycin,chloramphenicol or the like). For example, M13-series vectors,pUC-series vectors, pBR322, pBluescript, pCR-Script, etc. can be used.In addition, pGEM-T, pDIRECT and pT7 can also be used for subcloning andextracting cDNA as well as the vectors described above. When a vector isused to produce the protein of the present invention, an expressionvector is especially useful. For example, an expression vector to beexpressed in E. coli should have the above characteristics to beamplified in E. coli. When E. coli, such as JM109, DH5 alpha, HB101 orXL1 Blue, are used as a host cell, the vector should have a promoter,for example, lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEBJ 6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3(1988)), T7 promoter or the like, that can efficiently express thedesired gene in E. coli. In that respect, pGEX-5X-1 (Pharmacia),“QIAexpress system” (Qiagen), pEGFP and pET (in this case, the host ispreferably BL21 which expresses T7 RNA polymerase), for example, can beused instead of the above vectors. Additionally, the vector may alsocontain a signal sequence for peptide secretion. An exemplary signalsequence that directs the peptide to be secreted to the periplasm of theE. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379(1987)). Means for introducing of the vectors into the target host cellsinclude, for example, the calcium chloride method, and theelectroporation method.

In addition to E. coli, for example, expression vectors derived frommammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic AcidsRes 18(17): 5322 (1990)), pEF, pCDM8), expression vectors derived frominsect cells (for example, “Bac-to-BAC baculovirus expression system”(GIBCO BRL), pBacPAK8), expression vectors derived from plants (e.g.,pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV,pMV, pAdexLcw), expression vectors derived from retroviruses (e.g.,pZIpneo), expression vector derived from yeast (e.g., “Pichia ExpressionKit” (Invitrogen), pNV11, SP-Q01) and expression vectors derived fromBacillus subtilis (e.g., pPL608, pKTH50) can be used for producing thepolypeptide of the present invention.

In order to express the vector in animal cells, such as CHO, COS orNIH3T3 cells, the vector should have a promoter necessary for expressionin such cells, for example, the SV40 promoter (Mulligan et al., Nature277: 108 (1979)), the MMLV-LTR promoter, the EF1 alpha promoter(Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), the CMV promoterand the like, and preferably a marker gene for selecting transformants(for example, a drug resistance gene selected by a drug (e.g., neomycin,G418)). Examples of known vectors with these characteristics include,for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of thepresent invention.

EXAMPLES Materials and Methods

Cell Lines

T2, HLA-A*0201-positive B-lymphoblastoid cell line, and COS7, Africangreen monkey kidney cell line, were purchased from ATCC.

Candidate Selection of Peptides Derived from TOMM34

9-mer and 10-mer peptides derived from TOMM34 that bind to HLA-A*0201molecule were predicted using binding prediction software “BIMAS”(http://www-bimas.cit.nih.gov/molbio/hla_bind) (Parker et al. (J Immunol1994, 152(1): 163-75), Kuzushima et al. (Blood 2001, 98(6): 1872-81)).These peptides were synthesized by Biosynthesis (Lewisville, Tex.)according to a standard solid phase synthesis method and purified byreversed phase high performance liquid chromatography (HPLC). The purity(>90%) and the identity of the peptides were determined by analyticalHPLC and mass spectrometry analysis, respectively. Peptides weredissolved in dimethylsulfoxide at 20 mg/ml and stored at −80 degrees C.

In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as antigen-presentingcells to induce cytotoxic T lymphocyte (CTL) responses against peptidespresented on human leukocyte antigen (HLA). DCs were generated in vitroas described elsewhere (Nakahara S et al., Cancer Res 2003 Jul. 15,63(14): 4112-8). Specifically, peripheral blood mononuclear cellsisolated from a normal volunteer (HLA-A*0201 positive) by Ficoll-Paqueplus (Pharmacia) solution were separated by adherence to a plastictissue culture dish (Becton Dickinson) so as to enrich them as themonocyte fraction. The monocyte-enriched population was cultured in thepresence of 1000 U/ml of granulocyte-macrophage colony-stimulatingfactor (R&D System) and 1000 U/ml of interleukin (IL)-4 (R&D System) inAIM-V Medium (Invitrogen) containing 2% heat-inactivated autologousserum (AS). After 7 days of culture, the cytokine-induced DCs werepulsed with 20 micro-g/ml of each of the synthesized peptides in thepresence of 3 micro-g/ml of beta 2-microglobulin for 3 hr at 37 degreesC. in AIM-V Medium. The generated cells appeared to expressDC-associated molecules, such as CD80, CD83, CD86 and HLA class II, ontheir cell surfaces (data not shown). These peptide-pulsed DCs were theninactivated by X ray-irradiated (20 Gy) and mixed at a 1:20 ratio withautologous CD8+ T cells, obtained by positive selection with CD8Positive Isolation Kit (Dynal). These cultures were set up in 48-wellplates (Corning); each well contained 1.5×10⁴ peptide-pulsed DCs, 3×10⁵CD8+ T cells and 10 ng/ml of IL-7 (R&D System) in 0.5 ml of AIM-V/2% ASmedium. Three days later, these cultures were supplemented with IL-2(CHIRON) to a final concentration of 20 IU/ml. On day 7 and 14, the Tcells were further stimulated with the autologous peptide-pulsed DCs.The DCs were prepared each time by the same way described above. CTL wastested against peptide-pulsed T2 cells after the 3rd round of peptidestimulation on day 21 (Tanaka H et al., Br J Cancer 2001 Jan. 5, 84(1):94-9; Umano Y et al., Br J Cancer 2001 Apr. 20, 84(8): 1052-7; Uchida Net al., Clin Cancer Res 2004 Dec. 15, 10(24): 8577-86; Suda T et al.,Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005August, 96(8): 498-506).

CTL Expansion Procedure

CTLs were expanded in culture using the method similar to the onedescribed by Riddell et al. (Walter E A et al., N Engl J Med 1995 Oct.19, 333(16): 1038-44; Riddell S R et al., Nat Med 1996 February, 2(2):216-23). A total of 5×10⁴ CTLs were suspended in 25 ml of AIM-V/5% ASmedium with 2 kinds of human B-lymphoblastoid cell lines, inactivated byMitomycin C, in the presence of 40 ng/ml of anti-CD3 monoclonal antibody(Pharmingen). One day after initiating the cultures, 120 IU/ml of IL-2were added to the cultures. The cultures were fed with fresh AIM-V/5% ASmedium containing 30 TU/ml of IL-2 on days 5, 8 and 11 (Tanaka H et al.,Br J Cancer 2001 Jan. 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001Apr. 20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004 Dec. 15,10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9;Watanabe T et al., Cancer Sci 2005 August, 96(8): 498-506).

Establishment of CTL Clones

The dilutions were made to have 0.3, 1, and 3 CTLs/well in 96round-bottomed micro titer plate (Nalge Nunc International). CTLs werecultured with 1×10⁴ cells/well of 2 kinds of human B-lymphoblastoid celllines, 30 ng/ml of anti-CD3 antibody, and 125 U/ml of IL-2 in a total of150 micro-l/well of AIM-V Medium containing 5% AS. 50 micro-l/well ofIL-2 were added to the medium 10 days later so to reach a finalconcentration of 125 U/ml IL-2. CTL activity was tested on the 14th day,and CTL clones were expanded using the same method as described above(Uchida N et al., Clin Cancer Res 2004 Dec. 15, 10(24): 8577-86; Suda Tet al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci2005 August, 96(8): 498-506).

Specific CTL Activity

To examine specific CTL activity, IFN-gamma ELISPOT assay and IFN-gammaELISA were performed. Peptide-pulsed T2 (1×10⁴/well) was prepared asstimulator cells. Cultured cells in 48 wells were used as respondercells. IFN-gamma ELISPOT assay and IFN-gamma ELISA assay were performedunder manufacture procedure.

Establishment of the Cells Forcibly Expressing Either or Both of theTarget Gene and HLA-A02

The cDNA encoding an open reading frame of target genes or HLA-A*0201was amplified by PCR. The PCR-amplified product was cloned intoexpression vector. The plasmids were transfected into COS7, which is thetarget genes and HLA-A*0201-null cell line, using lipofectamine 2000(Invitrogen) according to the manufacturer's recommended procedures.After 2 days from transfection, the transfected cells were harvestedwith versene (Invitrogen) and used as the stimulator cells (5×10⁴cells/well) for CTL activity assay.

Results 1

Enhanced TOMM34 Expression in Cancers

The wide gene expression profile data obtained from various cancersusing cDNA-microarray revealed that TOMM34 (GenBank Accession No.NM_006809; for example, SEQ ID No: 42) expression was elevated. TOMM34expression was validly elevated in 4 out of 28 AMLs, 4 out of 14 CMLs, 8out of 11 bladder cancers, 1 out of 4 breast cancers, 1 out of 5cervical cancers, 12 out of 12 colorectal cancers, 5 out of 17 esophaguscancers, 6 out of 6 liver cancers, 1 out of 10 osteosarcoma, 1 out of 26prostate cancers, 1 out of 19 renal carcinomas, 2 out of 14 SCLCs, 5 outof 20 NSCLCs and 10 out of 51 soft tissue tumors as compared withcorresponding normal tissues (Table 1).

TABLE 1 Ratio of cases observed up-regulation of TOMM34 in canceroustissue as compared with normal corresponding tissue Cancer Ratio AML4/28 CML 4/14 Bladder Cancer 8/11 Breast Cancer 1/4  Cervical Cancer1/5  Colorectal Cancer 12/12  Esophagus Cancer 5/17 Liver Cancer 6/6 Osteosarcoma 1/10 Prostate Cancer 1/26 Renal Carcinoma 1/19 SCLC 2/14NSCLC 5/20 Soft Tissue Tumor 10/51 

Results 2

Prediction of HLA-A02 Binding Peptides Derived from TOMM34

Table 2a and 2b show the HLA-A02 binding 9mer and 10mer peptides ofTOMM34 in the order of high binding affinity. A total of 40 peptideswith potential HLA-A02 binding ability were selected and examined todetermine the epitope peptides.

TABLE 2a HLA-A02 binding 9mer peptides derived from TOMM34 Start SEQPosition amino acid sequence score ID NO 30 ALYGRALRV 222.566 1 77ALALVPFSI 60.51 2 52 VLYSNRAAC 27.026 3 110 TVLQIDDNV 11.034 4 220LLCSNLESA 9.518 5 230 YSNRALCYL 8.115 6 103 MAYVDYKTV 7.883 7 80LVPFSIKPL 7.309 8 255 KLDGKNVKA 6.955 9 23 GQYAEASAL 6.931 10 195VLKEEGNEL 5.211 11 111 VLQIDDNVT 5.194 12 238 LVLKQYTEA 4.101 13 1MAPKFPDSV 3.058 14 113 QIDDNVTSA 2.577 15 253 ALKLDGKNV 2.434 16 239VLKQYTEAV 2.028 17 144 LPSIPLVPV 1.775 18 142 LKLPSIPLV 1.398 19 279SSFADISNL 1.187 20

TABLE 2b HLA-A02 binding 10mer peptides derived from TOMM34 Start SEQPosition amino acid sequence score ID NO 143 KLPSiPLVPV 559.894 21 97ALEKyPMAYV 56.309 22 79 ALVPfSIKPL 49.134 23 237 YLVLkQYTEA 34.279 24135 SLGPeWRLKL 21.362 25 219 SLLCsNLESA 20.716 26 238 LVLKqYTEAV 18.75727 127 RMTRaLMDSL 17.388 28 113 QIDDnVTSAV 15.684 29 241 KQYTeAVKDC12.975 30 30 ALYGrALRVL 12.893 31 220 LLCSnLESAT 12.668 32 195VLKEeGNELV 8.314 33 112 LQIDdNVTSA 8.075 34 194 RVLKeEGNEL 6.916 35 299KLRQeVKQNL 5.682 36 141 RLKLpSIPLV 5.599 37 160 SLPSeNHKEM 4.968 38 175KETTaTKNRV 4.733 39 186 SAGDvEKARV 3.961 40

-   -   Start position indicates the number of amino acid residue from        the N-terminus of TOMM34.

Binding score is derived from “BIMAS”.

CTL Induction with the Predicted Peptides from TOMM34 Restricted withHLA-A*0201

CTLs for those peptides derived from TOMM34 were generated according tothe protocols as described in “Materials and Methods”. Peptide specificCTL activity was detected by IFN-gamma ELISPOT assay (FIG. 1a-d ). Thefollowing well numbers demonstrated potent IFN-gamma production ascompared to the control wells: well number #4 with TOMM34-A02-9-30 (SEQID NO: 1) (a), #2 with TOMM34-A02-9-220 (SEQ ID NO: 5) (b), #4 withTOMM34-A02-10-30 (SEQ ID NO: 31) (c) and #2 with TOMM34-A02-10-220 (SEQID NO: 32) (d). On the other hand, no specific CTL activity was detectedby stimulation with other peptides shown in Table 2a and 2b, despitethose peptides had possible binding activity with HLA-A*0201. As atypical case of negative data, specific IFN-gamma production was notobserved from the CTL stimulated with TOMM34-A02-10-143 (SEQ ID NO: 21)(e). As a result, it indicated that 4 peptides derived from TOMM34 wereselected as the peptides that could induce potent CTLs.

Establishment of CTL Line and Clone Against TOMM34 Derived Peptide

The cells that showed peptide specific CTL activity detected byIFN-gamma ELISPOT assay in the well number #4 with TOMM34-A02-9-30 (SEQID NO: 1) was expanded and CTL line was established by expansionprocedure as described in the “Materials and Methods” section above. CTLactivity of this CTL line was measured by IFN-gamma ELISA assay (FIG.2). The CTL line demonstrated potent IFN-gamma production against thetarget cells pulsed with the corresponding peptide as compared to targetcells without peptide pulse. Furthermore, the CTL clone was establishedby limiting dilution from the CTL line as described in “Materials andMethods”, and IFN-gamma production from the CTL clone against targetcells pulsed peptide was measured by IFN-gamma ELISA assay. PotentIFN-gamma production was observed from the CTL clone stimulated withTOMM34-A02-9-30 (SEQ ID NO: 1) (FIG. 3).

Specific CTL Activity Against Target Cells Expressing TOMM34 andHLA-A*0201

The established CTL line raised against TOMM34-A02-9-30 (SEQ ID NO: 1)peptide was examined for the ability to recognize target cells thatexpress TOMM34 and HLA-A*0201 molecule. COS7 cells transfected with boththe full length of TOMM34 and HLA-A*0201 gene (a specific model for thetarget cells that express TOMM34 and HLA-A*0201 gene) were prepared asstimulator cells, and COS7 cells transfected with either full length ofTOMM34 or HLA-A*0201 were used as the controls. In FIG. 4, the CTL linestimulated with TOMM34-A02-9-30 (SEQ ID NO: 1) showed potent CTLactivity against COS7 cells expressing both TOMM34 and HLA-A*0201. Onthe other hand, no significant specific CTL activity was detectedagainst the controls. Thus, these data clearly demonstrated thatTOMM34-A02-9-30 (SEQ ID NO: 1) peptide was endogenously processed andpresented on the target cells with HLA-A*0201 molecule and wasrecognized by the CTLs. These results indicate that this peptide derivedfrom TOMM34 may be suitable as a cancer vaccine for patients with TOMM34expressing tumors.

Homology Analysis of Antigen Peptides

The CTLs stimulated with TOMM34-A02-9-30 (SEQ ID NO: 1),TOMM34-A02-9-220 (SEQ ID NO: 5), TOMM34-A02-10-30 (SEQ ID NO: 31) andTOMM34-A02-10-220 (SEQ ID NO: 32) showed significant and specific CTLactivity. This result may be due to the fact that the sequence ofTOMM34-A02-9-30 (SEQ ID NO: 1), TOMM34-A02-9-220 (SEQ ID NO: 5),TOMM34-A02-10-30 (SEQ ID NO: 31) and TOMM34-A02-10-220 (SEQ ID NO: 32)are homologous to peptide derived from other molecules that are known tosensitize the human immune system. To exclude this possibility, homologyanalyses were performed for these peptide sequences using as queries theBLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) whichrevealed no sequence with significant homology. The results of homologyanalyses indicate that the sequence of TOMM34-A02-9-30 (SEQ ID NO: 1),TOMM34-A02-9-220 (SEQ ID NO: 5), TOMM34-A02-10-30 (SEQ ID NO: 31) andTOMM34-A02-10-220 (SEQ ID NO: 32) are unique and thus, there is littlepossibility, to our best knowledge, that this molecules raise unintendedimmunologic response to some unrelated molecule. In conclusion, weidentified novel HLA-A*0201 epitope peptides derived from TOMM34.Furthermore, the results herein demonstrate that epitope peptide ofTOMM34 may be suitable for use in cancer immunotherapy.

INDUSTRIAL APPLICABILITY

The present invention provides new TAAs, particularly those derived fromTOMM34 that induce potent and specific anti-tumor immune responses andhave applicability to a wide array of cancer types. Such TAAs are usefulas peptide vaccines against diseases associated with TOMM34, e.g.,cancer, more particularly, AML, CML, bladder cancer, breast cancer,cervical cancer, colorectal cancer, esophagus cancer, liver cancer,osteosarcoma, prostate cancer, renal carcinoma, SCLC, NSCLC and, softtissue tumor.

While the present invention is herein described in detail and withreference to specific embodiments thereof, it is to be understood thatthe foregoing description is exemplary and explanatory in nature and isintended to illustrate the present invention and its preferredembodiments. Through routine experimentation, one skilled in the artwill readily recognize that various changes and modifications can bemade therein without departing from the spirit and scope of the presentinvention, the metes and bounds of which are defined by the appendedclaims.

The invention claimed is:
 1. A method for inducing an antigen-presentingcell (APC) with CTL inducibility, wherein the method comprises a stepselected from the group consisting of: (a) collecting APCs from asubject, and (b) contacting an APC with a peptide in vitro, ex vivo orin vivo wherein the peptide is selected from the group consisting of: anisolated peptide of less than 15 amino acids comprising an amino acidsequence as shown in SEQ ID NO: 5, and an isolated peptide of less than15 amino acids comprising an amino acid sequence in which one or twoamino acid(s) are substituted, deleted, inserted or added to an aminoacid sequence as shown in SEQ ID NO: 5 to yield a modified peptide thatretains the ability to bind to an HLA antigen and cytotoxic T lymphocyte(CTL) inducibility.
 2. A method for inducing a CTL, comprising a stepselected from the group consisting of: (a) co-culturing a CD8-positive Tcell with an APC that presents on its surface a complex of an HLAantigen and a peptide, and (b) co-culturing a CD8-positive T cell withan exosome that presents on its surface a complex of an HLA antigen andthe peptide wherein the peptide is selected from the group consistingof: an isolated peptide of less than 15 amino acids comprising an aminoacid sequence as shown in SEQ ID NO: 5, and an isolated peptide of lessthan 15 amino acids comprising an amino acid sequence in which one ortwo amino acid(s) are substituted, deleted, inserted or added to anamino acid sequence as shown in SEQ ID NO: 5 to yield a modified peptidethat retains the ability to bind to an HLA antigen and cytotoxic Tlymphocyte (CTL) inducibility.
 3. A method of inducing an immuneresponse against cancer in a subject, wherein the method comprises astep of administering to the subject a peptide, or an immunologicallyactive fragment thereof, wherein the peptide is selected from the groupconsisting of: an isolated peptide of less than 15 amino acidscomprising an amino acid sequence as shown in SEQ ID NO: 5, and anisolated peptide of less than 15 amino acids comprising an amino acidsequence in which one or two amino acid(s) are substituted, deleted,inserted or added to an amino acid sequence as shown in SEQ ID NO: 5 toyield a modified peptide that retains the ability to bind to an HLAantigen and cytotoxic T lymphocyte (CTL) inducibility.
 4. The method ofclaim 1, wherein the peptide consists of the amino acid sequence of SEQID NO:
 5. 5. The method of claim 1, wherein the peptide has one or bothof the following characteristics: (a) the second amino acid from theN-terminus of the amino acid sequence of SEQ ID NO: 5 is substitutedwith methionine; and (b) the C-terminal amino acid of the amino acidsequence of SEQ ID NO: 5 is substituted with valine or leucine.
 6. Themethod of claim 2, wherein the peptide consists of the amino acidsequence of SEQ ID NO:
 5. 7. The method of claim 2, wherein the peptidehas one or both of the following characteristics: (a) the second aminoacid from the N-terminus of the amino acid sequence of SEQ ID NO: 5 issubstituted with methionine; and (b) the C-terminal amino acid of theamino acid sequence of SEQ ID NO: 5 is substituted with valine orleucine.
 8. The method of claim 3, wherein the peptide consists of theamino acid sequence of SEQ ID NO:
 5. 9. The method of claim 3, whereinthe peptide has one or both of the following characteristics: (a) thesecond amino acid from the N-terminus of the amino acid sequence of SEQID NO: 5 is substituted with methionine; and (b) the C-terminal aminoacid of the amino acid sequence of SEQ ID NO: 5 is substituted withvaline or leucine.